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In order to identify HIV-1 subtypes circulating in Tunisia, blood specimens from 25 HIV-1 infected Tunisian patients were collected. Proviral HIV-1 DNA was genotyped by sequence analysis of the C2-V3 env region. HIV-1 subtypes were determined in 21 DNA sequences: 20 were of subtype B and one was a circulating recombinant form (CRF02 AG). Subtype B largely dominates the epidemiology of HIV-1 infection in Tunisia, suggesting the probable imported origin of HIV-1 infection, but further studies are needed to investigate the potential diversification of HIV-1 isolates in Tunisia.