Human polyomavirus BK (BKV) and JC (JCV) infections were examined in persons infected with human immunodeficiency virus type 1 (HIV-1). High frequencies of BKV (24%) and JCV viruria (16%) were detected by polymerase chain reaction (PCR). BKV viruria was not found in an immunocompetent control group, in contrast to a frequency of JCV viruria of 20%. The degree of HIV-induced immunodeficiency did not influence the prevalence of BKV viruria, in contrast to cytomegalovirus viruria, suggesting BKV reactivation is an early manifestation in HIV infection as well as a temporal sequence of opportunistic infections. BKV DNA but not JCV DNA was detected in peripheral blood mononuclear cells (PBMC) in 2 of 42 subjects by a sensitive nested PCR. Sequencing of viral noncoding control regions (NCCRs) revealed predominantly archetypal and TU type BKV NCCRs but only archetypal JCV NCCRs. A new, naturally occurring BKV NCCR variant was detected in 1 urine specimen and 2 PBMC samples, indicating a stable and biologically significant rearrangement. Serum levels of BKV antibodies do not seem to be diagnostically useful in HIV-infected persons.