Development of an effective vaccine against HIV has faced great challenges due to the great genetic diversity of the virus. However, in different geographical areas candidate vaccines based on the prevalent HIV-1 subtype(s) are now entering clinical trials. Genotypic and serological assays have been used in HIV-1 subtyping. The peptide-binding enzyme immunoassay has been a useful tool for subtyping HIV-1. This assay in combination with an antigen limiting dilution assay was established at the Kilimanjaro Christian Medical Centre (KCMC) in Moshi, Tanzania. HIV-1 positive serum samples stored at KCMC were characterized using these assays to determine the trend since the start of the epidemic. In addition, the distribution of HIV-1 subtypes currently circulating in northern Tanzania was determined in order to provide important information that might be of relevance to future vaccine studies and trials in Tanzania.Frozen HIV-1 positive serum samples from individuals diagnosed at KCMC between 1985 and 2001 were used in this study. Synthetic peptides representing HIV-1 subtypes A, B, C, D and E derived from consensus gp120 V3 sequences were used in an indirect peptide-binding enzyme immunoassay and an antigen limiting dilution assay. The gp41 peptide D was used to increase the specificity by discriminating subtype D from non-D viruses. Two hundred and twenty six samples were analyzed; 196 (87%) samples were successfully subtyped while 30 (13%) could not be typed using these methods. In 1985 the prevalent subtypes were A (52%) and D (48%). Subtype C started to circulate in the late 1980s and in 2001 was the most prevalent circulating subtype in Tanzania. The currently circulating subtypes are A (32%), C (51%) and D (17%). HIV-1 vaccines entering clinical trials in Tanzania should be based on the predominant circulating subtype(s).