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Rio will pay A$25,000 for an exclusive six-month option over the non-gold minerals at the project.
It can then elect to sole-fund $5 million to earn 80% of the non-gold rights.
"We are delighted to be partnering with Rio Tinto Exploration to advance exploration for non-gold minerals within the northern part of our Rover project in Western Australia, complementing TSC's existing gold exploration activities," Twenty Seven CEO Simon Phillips said.
"The Yilgarn region is currently undergoing an exploration resurgence and the fact that we have attracted a global miner such as Rio Tinto demonstrates the underlying potential of our tenements. We look forward to providing updates on exploration progress in due course."
The Rover project comprises three exploration licences covering 460sq.km.
Twenty Seven has been exploring the ground for gold with recent holes returning low grades, but generating further targets.
In its December quarter report, the company said one hole had hit 4m of massive sulphides, within a 7m zone of disseminated sulphides from shallow depths.
Shares in Twenty Seven rose 12.5% to 0.4c.
