Operating from Te Anau Airport, five helicopters from Taieri company HeliOtago carried out the operation over 11,200ha in the valley.
Subject to suitable weather, it will be followed by a drop of 1080-laced pellets in a few days.
It is the first of at least 22 operations to drop 1080 over 600,000ha of South Island conservation land in the next three months.
It is aimed at knocking back rising numbers of rats and stoats caused by an unusually heavy seeding in beech forests known as a "beech mast".
The first stage of a similar operation in Southland's Waitutu Forest is expected to start today.
A lone protester was yesterday issued with a trespass notice by a Doc officer.
Professional hunter and guide Dave Wilson, of Te Anau, spent seven hours at the airport's entrance gate under the close watch of police and Doc staff.
Mr Wilson said he was protesting at the "inhumanity of the poison".
Doc science adviser Dr James Reardon said the Iris Burn operation was the first because it "couldn't wait any longer".
"We're very concerned about the number of rats in there already, and we're particularly worried about the bat population."
A remnant population of long-tailed bats discovered in the valley three years ago was "on a knife-edge".
The operation would also help protect whio, mohua, kiwi, kea and kaka.
The "pre-feeding" of non-toxic pellets -- "it's like a breakfast cereal" -- encouraged rats to eat the poison-laced pellets dropped later, Dr Reardon said.
