The second American was involved in an illegal hunt of a lion in April around Zimbabwe's Hwange National Park, said the National Parks and Wildlife Management Authority in a statement.
A Zimbabwean landowner named Headman Sibanda was arrested in the case and is assisting police with their investigation, the authority said.
Zimbabwean authorities earlier said they are seeking the extradition of Minnesota dentist Walter James Palmer, alleging he did not have authorisation to kill the lion named Cecil a month ago.
Palmer has said he relied on his professional guides to ensure the hunt was legal.
Two Zimbabwean citizens were arrested and face charges in the case in which Palmer has been implicated.
Yesterday, Zimbabwe's wildlife authority said it had suspended the hunting of lions, leopards and elephants in the Hwange area. Bow and arrow hunts were also suspended and can only be approved by the head of the wildlife authority.
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