The frequency of human T-cell lymphotropic virus types I and II (HTLV-I/II) polymerase chain reaction (PCR) reactivity was studied in two groups of high-risk individuals in Amsterdam: hard drug users and heterosexual outpatients of the sexual transmitted diseases (STD) clinic. Both groups were seronegative as determined by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), and radioimmuno-precipitation assay (RIPA). Detection of HTLV-I and HTLV-II in peripheral blood mononuclear cells (MNC) was performed by PCR, using primer sets indicative for the pol and tax genes. In the hard drug users group (n = 25) no evidence of HTLV-I/II infection was found whereas in the STD group (n = 21) one individual was identified with HTLV-II proviral DNA. Positive reactions in PCR were confirmed only for three seropositive controls after in vitro culture of MNC but not for the PCR-positive, seronegative individual. Virus production in vitro could not be detected by a sensitive HTLV-I antigen capture assay for viral p24gag proteins after in vitro T-cell stimulation of MNC, either from PCR-positive or PCR-seronegative individuals. This suggests again a low viral production rate. It is concluded that infection with HTLV-II can be detected among high-risk seronegative individuals.