Simian virus 40 (SV40), a small DNA tumor virus, was inadvertently administered to human populations with the use of contaminated vaccines. SV40 sequences have mainly been detected in healthy individuals and cancer patients using polymerase chain reaction techniques. However, some studies have failed to reveal the presence of SV40 in human specimens. These conflicting results indicate the need for new research to verify whether SV40 is circulating in humans. Mimotopes from SV40 structural peptides were tested to investigate for specific reactions to human sera antibodies. An indirect enzyme-linked immunosorbent assay with synthetic peptides from SV40 viral capsid proteins 1-2-3 (VPs 1-2-3) was set up and employed to test 855 serum samples from healthy blood donors. Data from immunologic assays indicate that serum antibodies against SV40 VP mimotopes are detectable, although with a low titer, in blood donors 18 to 65 years old. The overall prevalence of serum samples that reacted with the 2 SV40 VP peptides was 18%. The strong points for this novel method include the simplicity of its approach and the potential to discriminate between SV40-specific antibody responses and to draw correlations between responses to the 2 independent SV40 peptides. These data suggest that SV40, or a yet undetected closely related polyomavirus, is circulating in human populations, but with lower prevalence than that of the ubiquitous BK and JC human polyomaviruses.