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A cross-sectional serosurvey for human immunodeficiency virus type 1 (HIV-1) was conducted during the first quarter of 1991 among high risk groups in Djibouti, East Africa, and compared with previous surveys in 1987, 1988, and 1990. The survey demonstrated evidence of HIV-1 infection in 36.0% (n = 292) of street prostitutes, 15.3% (n = 360) of prostitutes working as bar hostesses, and 10.4% (n = 193) of males diagnosed with a sexually transmitted disease. By multivariate modeling, HIV-1 seropositivity in prostitutes was associated with Ethiopian nationality, working as a street prostitute, and residing in Djibouti for two years or less. We suggest that prostitution, particularly street prostitution, is a major route of HIV-1 transmission in Djibouti.