Detection of human T lymphotropic virus type I (HTLV-I) antibody was assessed on 368 sera from subjects with different clinical features and from different parts of the world. Enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay for purified p24 antibodies (p24-RIA) used as screening tests agreed in 88.7% of the sera. The results from 247 selected sera were compared with western blot (WB). WB was reactive in sera five to 25 times more dilute than the last positive ELISA or p24-RIA, but different WB batches varied in sensitivity. ELISA was more sensitive than p24-RIA, and p24-RIA was more specific than ELISA. Indeterminate WB interpretations were common (25.5%). Most seropositive intravenous drug abusers had unusually strong p24 bands by WB. Among healthy individuals, positive WB reactivity increased with age, whereas indeterminate reactivity declined (P = .034). Thus, more-sensitive and -specific HTLV-I antibody tests are needed.