Seropositivity to human T-cell lymphotrophic virus-III (HTLV-III) can have profound implications for the individual in whom it is detected. Simple and reliable tests are needed to confirm positivity by screening assays. In this study, detection of human antibodies by indirect immunofluorescence (IFA) on acetone-fixed HTLV-III infected H9 cells was evaluated in blood donors, patients with infectious or auto-immune diseases, and men with or at high risk for developing acquired immune deficiency syndrome (AIDS). Specific and nonspecific patterns of immunofluorescent reactivity were distinguished easily. None of 98 serums from blood donors was positive, while two of 33 serums from patients attending an infectious disease clinic, both homosexual men, were positive. Ninety-six percent of 24 serums from men with AIDS, 87 percent of 70 serums from men with lymphadenopathy, and 50 percent of 135 serums from healthy gay men were positive. These results paralleled those obtained by Western blotting and membrane immunofluorescence. In contrast, 11 and 4 percent, respectively, of these serums were judged as borderline or not interpretable by an enzyme-linked immunosorbent assay (ELISA). Of these serums, those that were positive by IFA were positive by Western blots, and 16 of the 17 IFA-negative serums were negative by Western blots. These studies indicate that IFA is a sensitive and specific assay for HTLV-III antibodies in human serums.