Natural transmission of human immunodeficiency virus (HIV) occurs at mucosal surfaces. During acute infection, intestinal and other mucosae are preferential sites of virus replication and rapidly become depleted of CD4(+) T cells. Therefore, mucosal immunity may be critical to control both initial infection and the massive early spread of virus. An attenuated D-alanine-requiring strain of the oral intracellular microorganism Listeria monocytogenes expressing HIV type 1 gag was shown to induce protective cell-mediated immunity in mice against viruses that express HIV gag when immunization occurs in the presence of a transient supply of D-alanine. In this study, we examined the efficacy of new attenuated strains that are able to synthesize d-alanine from a heterologous dal gene tightly regulated by an actA-promoted resolvase recombination system. In the absence of d-alanine, Gag-specific cytotoxic T lymphocytes (CTLs) were induced systemically after intravenous immunization, and one strain, Lmdd-gag/pARS, induced strong dose-dependent Gag-specific CTLs after oral immunization. A significant level of Gag-specific CD8(+) T cells was induced in the mucosal-associated lymphoid tissues (MALTs). Upon intravaginal challenge of these orally immunized mice with recombinant vaccinia virus (rVV) expressing HIV gag, gamma interferon- and tumor necrosis factor alpha-secreting Gag-specific CD8(+) T cells were dramatically increased in the spleen and MALTs. Oral immunization with Lmdd-gag/pARS led to complete protection against vaginal challenge by a homologous clade B gag-expressing rVV. In addition, strong cross-clade protection was seen against clades A and C and partial protection against clade G gag-expressing rVV. These results suggest that Lmdd-gag/pARS may be considered as a novel vaccine candidate for use against HIV/AIDS.