Vaccine protocols involving multiple immunizations with molecularly attenuated vaccinia virus (NYVAC) or naturally attenuated canarypox virus (ALVAC) HIV-2 recombinants and subunit boosts have conferred longlasting protection against HIV-2 infection of macaques. Similar complex protocols using HIV-1 NYVAC and ALVAC recombinants and subunit boosts have provided cross-protection against HIV-2 challenge. Here a simplified three-immunization regimen over 24 weeks was tested in 18 juvenile rhesus macaques. Twelve macaques were immunized twice with NYVAC or ALVAC recombinants carrying HIV-2 env, gag, and pol genes. Subsequently, macaques in groups of three received either an additional recombinant immunization or an HIV-2 gp160 boost. Six control macaques received three immunizations of NYVAC or ALVAC vector alone and additionally alum at the third immunization. Macaques primed with ALVAC recombinant exhibited sporadic T cell proliferative activity, and all but one failed to develop neutralizing antibodies. In contrast, macaques primed with NYVAC recombinants had no T cell proliferative activity but exhibited neutralizing antibody titers (highest in the three recombinant group) that declined by the time of challenge. None of the macaques exhibited significant cytotoxic T lymphocyte activity. Following challenge at 32 weeks with HIV-2SBL6669 all macaques became infected. Thus, the three-immunization regimen is not sufficient to confer protective immunity in the HIV-2 rhesus macaque model. However, delayed infection in macaques immunized with the NYVAC-HIV-2 recombinant may have been associated with the development of memory B cells capable of providing a neutralizing antibody response on challenge.