DNAs of the human polyomaviruses BK virus (BKV) and JC virus (JCV) were amplified by the polymerase chain reaction (PCR) by using a single pair of 20-base oligonucleotide primers that were complementary to the same regions of both viruses. The sequences flanked by the primers were unique for each virus and could be differentiated by hybridization with 40-base, 32P-labeled oligonucleotide probes or by cleavage with BamHI. The DNA fragments resulting from amplification of BKV and JCV were 176 and 173 nucleotide pairs, respectively. The sensitivities of PCR for amplification of cloned BKV and JCV DNAs were 10 and 100 copies, respectively. Hybridization with the oligonucleotide probes was specific for each virus. A total of 57 urine samples from three groups of subjects were processed by DNA extraction or boiling and were tested by PCR. Urine samples collected from immunosuppressed patients (n = 11) and previously documented to be positive for BKV, JCV, or both were positive by PCR. Ten percent of urine samples from healthy adults (n = 30) that were previously negative for BKV and JCV were positive for one or both viruses by PCR. Urine samples (n = 16) from four seronegative bone marrow transplant recipients were uniformly negative for BKV. JCV was detected in deparaffinized brain tissue from a patient with progressive multifocal leukoencephalopathy. Specific diagnosis of virus in clinical specimens could be made within 1 day of receipt of the specimens. The PCR method is attractive for use in diagnosing polyomavirus infections because of its sensitivity, specificity, and rapid turnaround time.