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In a joint petition for dissolution of marriage filed in King County Superior Court in Seattle, the couple stated: "The marriage is irretrievably broken."
But the foundation said Bill Gates (65) who co-founded Microsoft Corp, and Melinda Gates would continue to work together in their existing roles as co-chairs and trustees of the organisation.
The divorce filing, which states that the couple have no minor children, comes after the youngest of their three children is believed to have recently turned 18.
The spouses asked the court to approve their agreement on division of assets but did not disclose details.
"After a great deal of thought and a lot of work on our relationship, we have made the decision to end our marriage," the two said in a statement posted on each of their individual Twitter accounts.
"We no longer believe we can grow together as a couple in the next phase of our lives. We ask for space and privacy for our family as we begin to navigate this new life," they said.
Launched in 2000, the nonprofit Bill & Melinda Gates Foundation ranks as the largest private philanthropic foundation in the United States and one world's biggest, with net assets of $US43.3 billion ($NZ60.2 billion) at the end of in 2019, according to the latest full-year financials shown on its website.
From 1994 through 2018 Gates and his wife, who is 56, have provided gifts of more than $US36 billion to the Seattle-based foundation, the website said.
Last year, investor Warren Buffett reported donating more than $US2 billion of stock from his Berkshire Hathaway Inc to the Gates Foundation as part of previously announced plans to give away his entire fortune before his death.
The Gates Foundation has focused on public health, education and climate. Its initiatives include supporting development of coronavirus vaccines, diagnostic tests and medical treatments as well as support for public radio and the manufacture of solar-powered toilets.
JOINT FOUNDATION WORK TO CONTINUE
The pair "will continue to work together to shape and approve foundation strategies, advocate for the foundation’s issues and set the organisation’s overall direction," the Gates Foundation said in a statement.
The split comes two years after another leading American billionaire and philanthropist, Amazon.com Inc founder Jeff Bezos, said that he and his then-wife, MacKenzie, were getting divorced.
Gates dropped out of Harvard University to start Microsoft with school chum Paul Allen in 1975. Gates owned 49% of Microsoft at its initial public offering in 1986, which made him an instant multimillionaire.
With Microsoft's explosive growth, he soon became one of the world's wealthiest individuals.
After an executive tenure in which he helped transform the company into one of the world's leading technology firms, Gates stepped down as CEO of Microsoft in 2000 to focus on philanthropy.
He remained chairman until 2014 and left the company's board in March 2020.
Known in the technology industry as an acerbic and ruthless competitor, Gates drew the ire of rivals and eventually the US government for Microsoft's business practices.
The software giant was convicted of antitrust violations in the late 1990s. But the verdict was overturned on appeal, and the company then settled the case out of court.
Gates' public persona softened into an avuncular elder statesman as he turned his attention to philanthropy, and he has largely steered clear of the many controversies currently roiling the technology business.
Gates' spouse, who recently began referring to herself as Melinda French Gates on most websites and social media, was raised in Dallas and studied computer science and economics at Duke University.
She later joined Microsoft, where she met her future husband.
In 2015 she founded Pivotal Ventures, an investment company focused on women, and in 2019 published a book, The Moment of Lift, centred on female empowerment.