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Reports of human immunodeficiency virus (HIV) infection in seronegative individuals prompted the authors to investigate subjects who are at very high risk of acquisition of HIV in San Francisco. Nine HIV seronegative subjects were evaluated extensively, eight of whom were drawn from a well-characterized cohort of seropositive and seronegative homosexual men who have been followed prospectively since 1983-1984. These men have calculated probabilities of infection based on a fitted model of between 0.22 and 0.94. One additional subject is an intravenous drug user who has shared needles with HIV-infected individuals extensively. Peripheral blood lymphocytes and monocytes were separately cultured from each subject and evidence of HIV infection was sought by a reverse transcriptase assay, enzyme immunoassay, and immunocytofluorographic analysis for HIV antigens, in situ hybridization, RNA slot blot analysis, and polymerase chain reaction amplification of HIV cDNA. Uncultured monocytes and lymphocytes from each donor were also examined by these techniques. Evidence of HIV infection was not found in the peripheral blood mononuclear cells of these high risk individuals.