A series of recent studies have reported detection by the polymerase chain reaction (PCR) of cell-free human immunodeficiency virus type 1 (HIV-1) DNA (as opposed to virion RNA) in serum from both seropositive and seronegative persons. To evaluate the sensitivity, specificity, and reproducibility of PCR detection of cell-free HIV-1 DNA, we distributed coded panels containing 98 serum specimens obtained from well-characterized, infected individuals and control blood donors to the two laboratories with reported experience with this technique. Positive results were reported with HIV-1 gag primers (SK38/39) for 48 of 188 separate PCR determinations on DNA extracts from 44 serum samples from seropositive patients (25.5% sensitivity). HIV-1 gag signal was also reported for 28 of 151 PCR determinations on 34 samples from noninfected blood donors (18.5% false-positive rate). PCR for HIV-1 env DNA performed in one laboratory was negative on all specimens from seropositive and seronegative patients. Results for cell-free HIV-1 gag and human genomic (beta-globin or HLA DQ-alpha) DNA were inconsistent on replicate and serial specimens evaluated within each laboratory and between laboratories. These results indicate that current techniques for detecting cell-free HIV-1 DNA in serum lack adequate sensitivity, specificity, and reproducibility for widespread clinical applications.