To investigate the frequency of recombination between HIV-1 and HIV-2 in vivo during dual infection, we performed a retrospective analysis of blood samples from 46 dual HIV-1/HIV-2-seropositive adults for evidence of recombination. HIV viral DNA from peripheral blood mononuclear cells (PBMC) was subjected to two separate nested polymerase chain reaction (PCR) assays using opposing HIV-1 and HIV-2 primer pairs selected to flank a approximately 650-base pair region including the V3 loop of the envelope gene. In the first assay, primers were chosen to amplify recombinants with HIV-1 on the 5' end and HIV-2 on the 3' end, and in the second assay, primers were chosen to amplify recombinants with the opposite orientation. All PCR experiments were run in parallel with positive controls consisting of partial-length env fragments bearing a single central HIV-1/2 recombination site, and appropriate primer-binding sites on each end. The limit of detection for both assays was <10 copies of recombinant product per 150,000 cell equivalents of input PBMC DNA. In all 46 dually seropositive patients in this study, PCR screening of PBMC failed to detect evidence of HIV-1/HIV-2 recombinants in the C2-V5 env region. Although genetic recombination between HIV-1 and HIV-2 may occur, we conclude that any such events within env are exceedingly rare, and do not result in the outgrowth of recombinant strains.