Five female platypus have been released into the national park south of Sydney and four males will follow in the coming week after the quartet establish their territory.
The relocated platypus were collected from southern NSW to promote genetic diversity and brought to Sydney's Taronga Zoo, where they underwent veterinary health checks and were fitted with transmitters before release.
The Royal National Park - Australia's first official national park - has not been home to the native egg-laying mammals for 50 years after becoming locally extinct.
It is hoped the program, a collaboration between NSW National Parks and Wildlife Service, Taronga Conservation Society Australia, UNSW Sydney and World Wide Fund for Nature-Australia, will re-establish a self-sustaining and genetically diverse platypus population.
Platypus are under threat from habitat destruction and fragmentation amid increasing extreme climate events.
Taronga Conservation Society Australia's Cameron Kerr said the shy and enigmatic creatures were silent victims of climate change.
"While their elusive behaviour keeps them from view, under the surface they are particularly susceptible to drought and environmental change," he said.
"This translocation not only re-establishes a population in part of their former range but allows us to refine the skills and expertise that will inevitably be required to counter the impacts of increasingly frequent and more severe climate events."
WWF-Australia's rewilding program manager Rob Brewster believes the nation risks losing platypus forever if it doesn't take bold actions to reverse their decline.
"The last century saw the destruction of so much of Australia's wildlife and wild places," he said.
"The return of platypus to the Royal National Park shows that we can move beyond just protecting what remains, and actually restore what we've lost."
The former coalition NSW government quietly delayed the reintroduction program last year over water quality concerns after pollution events linked to the nearby Metropolitan mine at Helensburgh.
Dr Gilad Bino, from the UNSW's Centre for Ecosytem Science, said the project was also about restoring balance to the ecosystem.
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