We evaluated a new human immunodeficiency virus type 2 (HIV-2) DNA amplification strategy based on peripheral blood mononuclear cell long PCR (XL PCR) followed by nested PCR amplification. The primers used were located in the highly conserved long terminal repeat and in the pol regions of the genome. Five primer pairs corresponding to different regions of the HIV-2 env gene were used in the nested step. Samples from 42 patients were tested, which yielded positive amplification with at least two primer pairs in 40 (95%) samples. A primer pair (EB2/EB5) located on the V3 region succeeded in amplifying proviral DNA in 40 samples.