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The Ministry of Health (MoH) said this week some tenants would be leaving by the end of the year. The majority would leave early next year.
The MoH has bought the land the businesses are on, and plans to start demolition work early next year to start construction on the first stage of the hospital build, the smaller outpatient and day surgery building.
Businesses affected are Warehouse Stationery, Anytime Fitness and Lighting Direct, all on an access-way between Cumberland and Castle Sts.
The University of Otago is also moving from the site, although it has already organised new sites to occupy.
Anytime Fitness New Zealand marketing manager Craig Cawkwell, of Hamilton, said the company supported the new hospital.
But he understood no new site had yet been found for the business.
"I think it's quite challenging," he said of the search for a suitable site.
The national office was supporting Dunedin owner Bob Sanders to find a solution.
Asked whether Warehouse Stationery had found a new home, and if workers would keep their jobs, a Warehouse Group spokesman said the company was working through different options.
Lighting Direct did not respond to calls.
A university spokeswoman said Otago University Press would remain in its building until the end of its lease on March 31 next year. It would then relocate to 533 Castle St, a university-owned villa converted into offices near the Centre for Innovation.
Dentistry staff would go to the university-owned building at 71 Frederick St.
There has been no confirmation yet on the MoH's work to buy the Wilson Parking land on the corner of St Andrew and Cumberland Sts, but news of a sale was expected soon.
Wilson Parking would say only: "we confirm that we are committed to continuing to operate in Dunedin".