Current screening tests to detect human T-lymphotropic virus type I (HTLV-I) in volunteer blood donors commonly yield indeterminate HTLV serologic results (mostly isolated gag reactors). To assess the significance of indeterminate HTLV serologic results in U.S. blood donors, we compared 56 persons who had such serologic patterns with 30 HTLV seropositive blood donors and with HTLV seronegative controls. Polymerase chain reaction assays showed that none of the 56 individuals with indeterminate HTLV serologic results were infected with HTLV-I or HTLV-II, while all 30 HTLV seropositive blood donors were infected with either HTLV-I (in 15) or HTLV-II (in the other 15). The seroindeterminate blood donors were also different from the HTLV seropositive blood donors and more like HTLV seronegative controls in their demographic characteristics and the presence of HTLV risk factors. These results are evidence that volunteer blood donors with isolated and persistent gag seroreactivity in the United States are unlikely to be infected with HTLV-I or HTLV-II.