Summary:The objective of this study was to determine the frequency and significance of nondiagnostic Western blot (WB) assays in homosexual/bisexual men at risk of infection with HIV-1. The presence of a positive enzyme-linked antibody assay (EIA) confirmed by a positive WB was used as evidence of infection and seroconversion. Indeterminate WB assays were defined as reactions to only one viral gene product of HIV-1. Three analyses were conducted to (a) determine the frequency of such reactions in men who, during a 4-year period, did not develop diagnostic serologic reactions; (b) determine, retrospectively, the preseroconversion frequency of indeterminate WB assays in 286 men who seroconverted; and (c) evaluate in vitro production of specific antibody by peripheral blood mononuclear cells (PBMCs) as a method of indicating whether or not an indeterminate WB assay represents HIV-1 infection. Reactions to products ofgag, pol, or envwere noted in 8.0, 4.0, and 6.7% of 1,595 first-visit tests of men who remained seronegative for 4 years. Indeterminate reactions occurred in 204 men with negative EIAs who subsequently seroconverted and in 82 men with positive EIAs preconversion. Supernatants harvested from PBMCs of 2 of 36 seroconverters obtained one or two visits preseroconversion and cultured with pokeweed mitogen were antibody-positive. All were positive at the visit, with diagnostic serology. None of the supernatants from cells of 19 men with EIA-negative WB-indeterminate serologic assays were antibody-positive. Our results suggest that persistently EIA-negative homosexual/bisexual men who have indeterminate WB assays are unlikely to be infected with HIV-1. In contrast, EIA-positive, WB-indeterminate serologic assays in this cohort were frequently associated with subsequent seroconversion.