The groups argued that the federal government hasn't followed strict rules on when to allow landowners to kill wolves, and that even supposedly humane removals have contributed to deaths.
Some landowners have complained that the wolves cause problems when they stray onto private land.
The arguments concern current red wolf rules, but the case could also affect new plans by the federal government to sharply reduce red wolf territory. Federal Judge Terrence Boyle said he'd rule later on the preliminary injunction request.
About 45 red wolves roam wild in eastern North Carolina, less than half the peak population since they were reintroduced three decades ago.
Southern Environmental Law Center lawyer Sierra Weaver argued that the removal methods they're challenging will be central to the new plan to reduce the wolves' territory.
"Our concern is that they have decided to end this population, but they have avoided judicial review," she said. "Taking animals off the landscape and putting them in zoos is not consistent with the Endangered Species Act."
The hearing came two days after federal officials announced plans to reduce the wolves' territory starting in 2017 to a federal wildlife refuge and adjacent land in Dare County, rather than the wolves' current five-county territory. The new plan is contingent on modifying current program rules after a public comment period.
Wednesday's hearing had been previously scheduled on conservationists' arguments that the federal government twice gave landowners permission to kill wolves without meeting strict legal requirements since 2014. One wolf was shot as a result.
Justice Department lawyers representing the U.S. Fish and Wildlife Service argued that the conservationists are misinterpreting regulations that give federal officials wide leeway to remove animals humanely or authorize lethal means. They say that any removals are done after thorough reviews of each case. It's generally illegal to kill the wolves without permission.
Government lawyer Lesley Lawrence-Hammer argued that the conservation groups can't win emergency intervention because they've failed to show how they're harmed when wolves are removed from private land.
"What harms do plaintiffs face here?" she asked. "Plaintiffs have no right to see, hear or enjoy wolves on private land."
Once common around the Southeast, the red wolf had been considered extinct in the wild as of 1980 because of factors including hunting and habitat loss. Releases of red wolves bred in captivity started in 1987.
While Boyle isn't deciding directly on the new federal plan for the wolves, he noted that federal officials have proposed confining the wild population to a peninsula that frequently floods.
"You're pushing the wolves back into the least viable and least attractive land in this area," the judge told the government's lawyers.
Boyle previously gave environmentalists a win in a separate case when he temporarily halted coyote hunting in the wolves' territory, and he later approved a permanent agreement in 2014 banning nighttime coyote hunting in the five-county area. Conservationists say red wolves can easily be mistaken for coyotes by hunters.
The federal government has also argued that the 200 or so red wolves living in captivity justify the wild wolves' classification as a "nonessential" population.
Lawrence-Hammer told the judge on Wednesday that nonessential populations face different rules than other endangered species.
Boyle retorted that no matter how the wolves are classified now, "When they're all gone they will be an 'extinct species.'"