We developed an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to simian virus 40 (SV40) in human serum. We then used the ELISA to test for anti-SV40 antibodies in healthy inviduals and patients suffering from cancer and renal diseases. The aim of the study was to determine the presence of antibodies against SV40 in sera of individuals who received the South African poliovirus vaccines from 1958 to the present. Detecting such antibodies could give an indication of whether any of the poliovirus vaccines used in South Africa were ever contaminated with the SV40 or not. A total of 5/164 samples were repeatedly positive for SV40 antibodies by the ELISA. Four of the samples were from the healthy population group and the remaining 1 (1/64) was from the patient group. An SV40 antibody-blocking assay and a Western blot were used as additional confirmation for the SV40 antibodies, whereas the Western blot assay developed a single common band on all 5 samples