Feline immunodeficiency virus (FIV) is a novel lentivirus that causes T-cell deficiency in the domestic cat (Felis catus). Recent studies have revealed the existence of antigenically similar lentiviruses in a large number of nondomestic felid species. We summarize here a comprehensive serological survey for FIV cross-reactive antibodies in free-ranging and captive felid populations. Serum or plasma samples from 1,645 animals representing 20 felid species were screened by western blot analysis for exposure to one or more lentiviral proteins. Feline lentiviruses were confirmed to be endemic in several East and South African populations of lion (Panthera leo). Exposure was also detected in nearly all of the natural North American puma (Felis concolor) populations surveyed. Antibodies to FIV were also found in a free-ranging population of cheetah (Acinonyx jubatus). Lentivirus presence among captive exotic cats in the United States and abroad appears to be sporadic and infrequent. Because FIV is endemic in certain natural populations and absent in others, these data suggest that lentivirus infection postdated the geographic separation of felid populations; and therefore, spread of the virus into other seronegative populations may now be restricted by natural geographical barriers.