An enzyme-linked immunosorbent assay (ELISA) using a puma lentivirus-derived synthetic peptide as coating antigen was evaluated as a diagnostic test for infection with feline immunodeficiency virus (FIV) or related lentiviruses in free-ranging lions. The sensitivity and specificity of the ELISA was determined using two approaches. In the first approach, the results were standardized according to certain statistical criteria, and in the second, the puma lentivirus western blot was used as the gold standard. The sensitivity of the test when compared with the standardized results was 85.4% and the specificity 100%. The sensitivity of the test when using the western blot as the gold standard was 78.6% and the specificity 100%. The test would therefore be well-suited to the screening of populations of wild felids in which FIV or related lentiviruses are endemic. The results also indicate that in spite of genetic divergence between lentiviruses isolated from Panthera and Felis spp., puma lentivirus-derived antigens can be used in immunoassays for the detection of antibodies in Panthera spp. reactive to FIV or related lentiviruses. The results also indicate that the lion population in the Hluhluwe-Umfolozi Game Reserve, South Africa is lentivirus negative.