To confirm the presence of DNA from human T lymphotropic virus type I (HTLV-I), HTLV-II, or both in individuals found HTLV-I/II-positive through systematic screening of blood donations in Guadeloupe (French West Indies), 42 blood donors repeatedly positive for HTLV-I/II by ELISA were studied by polymerase chain reaction (PCR). Three primer pairs (env, pol, tax) targeted on conserved regions of HTLV-I or -II sequences (or both) and six probes (two generic, two HTLV-I-specific, two HTLV-II-specific) were used in a multiplex PCR. HTLV-I sequences were detected in 31 individuals (74%). All 31 subjects positive by Western blot (WB) harbored HTLV-I sequences. Fifteen individuals (48%) were positive with the three primer pairs used, 10 (32%) with two, and 6 (20%) with one. Subjects indeterminate or negative by WB were all negative by PCR. No HTLV-II sequences were detected with specific probes. The results indicate the absence of HTLV-I and -II infection in individuals with indeterminate WB, the presence of HTLV-I DNA in individuals positive for WB in the French West Indies, and the absence of HTLV-II infection in the cohort.