Listeria monocytogenes is a facultative intracellular bacterium that lives and grows in the cytoplasm of the host cell. The hallmark of a listerial infection is a cell-mediated immune response to its own secreted virulence factors. Thus, L. monocytogenes vaccines engineered to secrete HIV proteins may be ideal vectors for boosting cellular immune responses against HIV. Using strains of L. monocytogenes that stably express and secrete HIV Gag (Lm-Gag) to deliver this Ag to the immune system, we have previously shown strong MHC class I-restricted cytotoxic T cell responses to this protein. In this study, we examine MHC class II-restricted T cell responses to HIV-Gag delivered by Lm-Gag. We demonstrate the induction of CD4+ T cells that are HIV-Gag specific and identify three epitopes in two strains of mice, BALB/c (H-2d) and C57BL/6 (H-2b), two of which are both H-2d and H-2b restricted, but are not immunodominant for both haplotypes. In addition, we show that the CD4+ T cells induced are of the Th1 phenotype that produce IFN-gamma at levels similar to CD4+ T cells induced to endogenous listerial Ags. These studies suggest that chromosomally modified strains of L. monocytogenes may be useful as vaccine vectors for the induction of Th1 T cell responses against HIV.