The performance and clinical relevance of a qualitative PCR-based assay for the detection of HIV-1 DNA sequences in peripheral blood mononuclear cells (PBMCs) was evaluated by two different laboratories. Four hundred and one samples were obtained from 397 individuals from different risk populations. All blood donors tested had negative results; positive signals were obtained from all infected patients. HIV-1 DNA was detected in 3 of 17 infants born to seropositive mothers; Western blot indeterminate blood donors and exposed health-care workers had negative results. Our results demonstrate that this PCR assay provides both sensitive and specific results and is suitable for testing large numbers of samples and for rapid identification of HIV-1 infection.