To determine the presence or the absence of human T-lymphotropic virus type I and/or II (HTLV-I/II) DNA in at-risk individuals who were persistently negative for specific serologic assays, polymerase chain reaction with two primer pairs in common and conserved regions of HTLV-I and -II genomes was used. Seronegative individuals at risk for HTLV-I/II infection (15 heterosexual partners of seropositive individuals, 17 breastfed children born to HTLV-I-infected mothers, 47 multiply transfused patients, 22 intravenous drug users) were studied (n = 101); 35 seropositive individuals and 25 seronegative low-risk individuals were used as positive and negative controls, respectively. No positive polymerase chain reaction was observed in the seronegative at-risk individuals or in the negative controls. Positive controls gave positive results with at least one primer pair in all cases except one. A latent HTLV-I/II infection with a persistently negative serologic test for HTLV-I/II seems unlikely.