Two recombinant Listeria monocytogenes (rLm) strains were produced that secrete the human papilloma virus-16 (HPV-16) E7 protein expressed in HPV-16-associated cervical cancer cells. One, Lm-E7, expresses and secretes E7 protein, whereas a second, Lm-LLO-E7, secretes E7 as a fusion protein joined to a nonhemolytic listeriolysin O (LLO). Lm-LLO-E7, but not Lm-E7, induces the regression of the E7-expressing tumor, TC-1, established in syngeneic C57BL/6 mice. Both recombinant E7-expressing rLm vaccines induce measurable anti-E7 CTL responses that stain positively for H-2D b E7 tetramers. Depletion of the CD8+ T cell subset before treatment abrogates the ability of Lm-LLO-E7 to impact on tumor growth. In addition, the rLm strains induce markedly different CD4+ T cell subsets. Depletion of the CD4+ T cell subset considerably reduces the ability of Lm-LLO-E7 to eliminate established TC-1 tumors. Surprisingly, the reverse is the case for Lm-E7, which becomes an effective anti-tumor immunotherapeutic in mice lacking this T cell subset. Ab-mediated depletion of TGF-β and CD25+ cells improves the effectiveness of Lm-E7 treatment, suggesting that TGF-β and CD25+ cells are in part responsible for this suppressive response. CD4+ T cells from mice immunized with Lm-E7 are capable of suppressing the ability of Lm-LLO-E7 to induce the regression of TC-1 when transferred to tumor-bearing mice. These studies demonstrate the complexity of L. monocytogenes-mediated tumor immunotherapy targeting the human tumor Ag, HPV-16 E7.