Sequences from the nef/LTR overlap region of the human immunodeficiency virus type 2 (HIV-2) genome were amplified from uncultured peripheral blood mononuclear cells (PBMCs) from 40 HIV-2-infected individuals in The Gambia, West Africa. Additional sequences from the plasma of three blood donors were also derived. Analysis of HIV-2 U3 LTR transcription factor elements (PuB-1, p-ets, PuB-2, peri-kappa B, and NF-kappa B sites) indicated a relatively high level of conservation in vivo. The region immediately 3' of the nef termination codon, which exhibits clade-dependent specificity, was targeted by PCR to differentiate HIV-2 subtype A from subtype B infections, the two principal clinical HIV-2 subtypes. All clinical samples analyzed (n = 43) from The Gambia were identified as HIV-2 subtype A by a combination of LTR sequence analysis and subtype-specific amplification of subtypes A and B. Differential PCR amplification of the HIV-2 U3 LTR region represents a rapid means of differentiating subtype A from subtype B infections, the two dominant HIV-2 subtypes that are important in human disease.