Follow-up studies on 67 blood donors with indeterminant serological findings for human T-lymphotropic virus (HTLV) type I by standard immunoassays showed no evidence of infection by polymerase chain reaction analysis for HTLV-I or HTLV-II nucleic acids or by antibody reactivity to a unique HTLV-I recombinant envelope protein, MTA-4. Among HTLV-I- or -II-infected individuals, a history of blood transfusion, past residence in established HTLV-I endemic areas or some association with intravenous drug use were common. In contrast, 85% of indeterminant cases had none of these risk factors. These observations suggest that healthy individuals with indeterminant serology for HTLV-I should not require additional studies.