Forty-seven mountain lions (Puma concolor) collected year-round in 1996 to 1998 from the Sierra Nevada foothills, the northern coast ranges, and in Monterey County (California, USA) were examined for infestation with Ixodes pacificus and Dermacentor variabilis ticks. Ticks were found predominantly in winter and spring. The seroprevalence of granulocytic ehrlichiae (GE) antibodies (Ehrlichia equi or the agent of human granulocytic ehrlichiosis) was 17% and the PCR-prevalence of DNA characteristic of GE in blood was 16%. There were eight polymerase chain reaction (PCR)-positive but seronegative mountain lions, one that was PCR-positive and seropositive, and eight that were PCR-negative and seropositive. Nineteen percent of engorged tick pools from mountain lions were PCR-positive. Because mountain lions inhabit tick-infested habitat and are frequently bitten by I. pacificus, surveillance for GE antibodies and DNA in mountain lions and other vertebrate hosts may be useful as indicators for geographical regions in which humans are at risk of GE infection.