Elicitation of both memory cytotoxic T cell responses and human immunodeficiency virus (HIV)-neutralizing antibodies are desirable characteristics of an HIV vaccine regimen. We studied a combination vaccine regimen consisting of a canarypox (CP) vector containing env, gag, and pro, in combination with a recombinant gp120 subunit protein. Twenty-six of 42 subjects who received CP vac-env-pro demonstrated in vitro CD8+ T cell responses, versus 3 of 17 who received the control CP rabies or gp120 vaccine only (P = .0003); 15 of these 26 demonstrated a CD8+ cytotoxic T lymphocyte (CTL) response on ≥2 occasions postvaccination. The frequency of CD8+ CTL response to HIV antigens was similar between vaccinianaive and vaccinia-immune persons. Rgp120 immunization did not increase the CD8+ CTL response to HIV type 1 envelope proteins, but rgp120 boosting did markedly enhanced the titer and frequency of neutralizing antibodies to the MN strain of HIV. Overall, the combination of a CP gag, pro, env vector, in combination with recombinant gp120, resulted in neutralizing antibodies in 91% of subjects and CD8+ T cell responses in 62% of subjects. A nonreplicating pox virus shuttle vector vaccine appears to be capable of eliciting CD8+ CTL responses in most healthy volunteers, whether they were vaccinia naive or vaccinia immune.