Widely disseminated infection with the human polyomaviruses JC and BK is followed by lifelong asymptomatic viral persistence that can be reactivated under prolonged immunosuppression to fatal CNS and urogenital disease. In an attempt to understand the pathogenesis of polyomavirus diseases, we asked whether leukocytes are involved in polyomavirus infection in the immunocompetent host. Peripheral blood leukocytes from 29 immunocompetent individuals and umbilical cord blood from 10 newborn children were analyzed for the presence of polyomavirus DNA. Southern blot analysis demonstrated the presence of JCV-specific full-length virus genomes and indicated involvement of the second human polyomavirus BK. In contrast to specimens from newborn children, PCR amplification of target DNA in the adult age group followed by species-specific hybridization provided evidence of concomitant JCV and BKV infection in almost all specimens. Nucleotide sequencing of virus-specific products representing DNA segments essential for virus multiplication confirmed presence of both virus species in leukocytes. The detection of a new virus subtype and single base changes or deletions in the noncoding DNA region from individual cases suggested widespread heterogeneity in the circulating virus population, although the structure of the transcriptional control elements in all cases was comparable to highly active elements found in lytically infected cells. Those findings and the localization of both virus types in the nuclei of blood cells by in situ hybridization demonstrate that JCV and BKV frequently infect peripheral leukocytes and give strong evidence that leukocytes are common sites of polyomavirus persistence in healthy individuals.