Current tests to confirm human T-cell lymphotropic virus (HTLV) infection in individuals at risk of retroviral infection commonly yield indeterminate results. To assess the significance of HTLV-seroindeterminate reactivities in a high-risk population, 16 at-risk individuals who had this serologic pattern by Western blot were studied using a polymerase chain reaction (PCR) assay. Human T-cell lymphotropic virus type II infection was confirmed by the presence of virus-specific nucleic acid in four patients. However, PCR analysis was negative in the remaining 12 individuals. These results indicate strongly that all specimens from at-risk individuals with nondiagnostic HTLV reactivity by current Western blot assay should continue to be considered inconclusive, requiring further testing by more sensitive tests.