Burrowing owls nest and roost in ground squirrel burrows, a refuge frequently used by rattlesnakes. When cornered, burrowing owls produce a vocal hiss that has been suggested to mimic a rattlesnake's rattle. To test this hypothesis, we conducted an experiment using two populations of Douglas ground squirrels that differ in their evolutionary histories with rattlesnakes. Both squirrel populations were sympatric with burrowing owls. Squirrels from a population subjected to natural selection by rattlesnakes treated the owl hiss as cautiously as they did the rattle, and responded with greater caution to the rattle and hiss than to two control sounds. Squirrels from a rattlesnake-free area, however, were less systematic in differentiating among the rattle, the hiss, and the control treatments. Such variation between ground-squirrel populations provides evidence that the burrowing owl's defensive hiss currently functions as an acoustic Batesian mimic of a rattlesnake's rattle.