Puma lentivirus (PLV) antibodies were detected in 13 (25%) of 52 serum samples obtained from cougars (Felis concolor) collected by hunters. The serum samples were collected from November 1993 through January 1994 from four specific regions throughout the state of Washington (USA), and included the Olympic Mountains, the Cascade Mountains, the Blue Mountains, and the Selkirk Mountains. More (38%) seropositive cougar samples originated from the Cascade Mountains than from any other site. The overall seroprevalence for PLV infection in Washington cougars was higher than previously reported for cougars sampled in Oregon and Idaho (USA), but lower than in cougars sampled in Arizona, Colorado, and California (USA).