BACKGROUND: Reverse transcriptase (RT) is present in all infectious retrovirus particles. Sensitive RT tests should thus detect all such particles. A family of ultrasensitive RT tests, product-enhanced reverse transcriptase (PERT) assays, have been designed. Accumulated results show that (i) a first version of the PERT assay that uses microtiter/ELISA technology detects RT of only 3-11 retroviral particles, (ii) very different human and animal lenti- and oncoviruses are detected very sensitively, (iii) HIV-1 is detected as sensitively as with polymerase chain reaction (PCR) for viral RNA, and (iv) prevalence of elevated particle-associated RT in plasma of unselected Swiss blood donors was 1.9%. OBJECTIVE: To investigate whether the RT activity detected in one unselected donor with a chronically elevated level of the liver enzyme alanine aminotransferase and two selected donors with chronically indeterminate or borderline-positive HIV-1 serology was due to human immunodeficiency virus (HIV) or human T-cell leukemia virus (HTLV) infection. STUDY DESIGN: Blood samples were tested by PCR for viral DNA and/or RNA of HIV-1, HIV-2, HTLV-1, HTLV-2, hepatitis B and C virus. Serological tests and virus cultures were also employed. RESULTS: Infection with any of the above agents could not be demonstrated. Virus cultivation in one case of borderline-positive HIV-1 Western blot for more than 5 years yielded a peak of RT production that was repeatedly transmissible to fresh cells. CONCLUSIONS: The findings suggest the presence of an infectious RT-positive agent, probably different from HIV-1/2 or HTLV-1/2, in a healthy individual with chronically borderline-positive HIV-1 serology. The PERT assay may detect retroviruses currently undetectable by other tests. The use of more stringent Western blot interpretation guidelines other than those of CDC or WHO is strongly recommended.