Surveillance for HIV-1 prevalence and subtypes in Afar Region, Ethiopia was performed among police recruits in the year 2000, by unlinked anonymous testing. Of 408 samples tested, 26 (6.4%) appeared positive for HIV-1 antibodies. There was a trend for higher HIV-1 seroprevalence in women (9.5%, 9/95) than men (5.4%, 17/313), which was significant in one of the 5 administrative areas: Zone 4 (p = 0.01). Around the principal transportation route connecting Addis Ababa to the harbor of Djibouti there was a significantly (p = 0.03) higher HIV-1 seroprevalence of 12.7% (14/110) than elsewhere in Afar Region. In addition, 13 (34%) of the 29 administrative sub-areas (woredas) of Afar Region delivered HIV-1 positive police recruits. Prevalence of syphilis antibodies was 7.4% (30/408), increasing by age, correlating with HIV-1 positive serology (p = 0.001) and with 23.3% (7/30) active cases. Of 22 specimens sequenced, 12 had gp120 V3 regions from Ethiopian subtype C, 9 subtype C' and 1 subtype A. In conclusion, even in very remote areas in Ethiopia, such as Afar Region, the HIV-1 epidemic is established, being primarily of subtype C. Regular HIV-1 surveillances will be necessary to guide action to prevent further spread in this vulnerable area