Simian virus SV40, an oncogenic virus in rodents, was accidentally transmitted to humans through the Poliovirus vaccine during the years 1955 to 1963. If the vaccination program were the major source of human infection, then differences in SV40 infection rates by cohort of birth should be observed. The aim of this study was to address this issue. In 134 healthy Italian Caucasian subjects, 15 DNA samples were positive for SV40 by nested polymerase chain reaction and DNA sequencing. The prevalence of genomic infection did not differ across cohorts of birth from 1924 to 1983, however DNA sequencing revealed viral strain differences in individuals born before 1947 and after 1958. While horizontal transmission following the introduction of the polio vaccine could explain the presence of SV40 DNA in younger people, our results also suggest the possibility that other sources of the virus may also be involved in human SV40 infection.