Tests for the enzyme reverse transcriptase (RT) should permit the detection of all infectious retroviruses, provided that these are present as extracellular particles. The capability of a new procedure, named product‐enhanced reverse transcriptase (PERT) assay, to detect HIV‐1 in fresh human plasma was compared with that of the polymerase chain reaction (PCR) for viral RNA. Both procedures had identical dilution endpoints corresponding to 10² particles/ml. All 30 samples from HIV‐1 positive patients at different stages contained RT activity whose level was significantly correlated with viral RNA and corresponded to 553‐417,000 particles/ml. In HIV‐1 low titer performance and seroconversion panels, the PERT assay detected more positives than PCR for viral RNA. Three of 160 blood donors exhibited elevated RT activity, indicating a prevalence of 1.9% (95% CI 0.4–5.3%). One positive donor, with laboratory parameters suggesting a mild chronic liver impairment, exhibited RT activity comparable to that of HIV positives, but was consistently negative by various tests for hepatitis viruses, cytomegalovirus, the HIVs and HTLVs. The results suggest that the PERT assay is more sensitive for detection of HIV‐1 contamination of plasma than RNA PCR. However, it is not affected adversely by viral sequence variability, and may therefore, also detect HIV‐1 subtype O, and additional retroviruses as yet undetectable by PCR. © 1996 Wiley‐Liss, Inc.