An elevated number of Gr-1⁺CD11b⁺ myeloid cells has been described in mice bearing transplantable tumors, and has been associated with immune suppression. We examined the role of such myeloid suppressor cells in mice bearing the spontaneously transformed syngeneic mouse ovarian surface epithelial cell line, 1D8. We observed high levels of CD80 expression by Gr-1⁺CD11b⁺ cells from spleen, ascites, and tumor tissue of mice bearing 1D8 ovarian carcinoma, whereas CD40 and CD86 were absent. CD80 expression was not detected on Gr-1⁺CD11b⁺ cells from naïve mice. However, the expression of CD80 by Gr-1⁺CD11b⁺ cells from naïve mice was promoted by coculture with 1D8 cells. Because irradiated 1D8 cells, but not 1D8-conditioned medium, up-regulate CD80 expression by Gr-1⁺CD11b⁺ cells, this phenomenon likely requires direct interaction. Gr-1⁺CD11b⁺ cells derived from 1D8 tumor–bearing mice provided significant suppression of antigen-specific immune responses, but Gr-1⁺CD11b⁺ cells from naïve mice did not. Both short interfering RNA–mediated knockdown and genetic knockout of CD80 expression by Gr-1⁺CD11b⁺ cells of 1D8 tumor–bearing mice alleviated the suppression of antigen-specific immune responses. Suppression via CD80 on Gr-1⁺ CD11b⁺ myeloid cells was mediated by CD4⁺CD25⁺ T regulatory cells and required CD152. CD80 knockout or antibody blockade of either CD80 or CD152 retarded the growth of 1D8 tumor in mice, suggesting that expression of CD80 on Gr-1⁺CD11b⁺ myeloid cells triggered by 1D8 ovarian carcinoma suppresses antigen-specific immunity via CD152 signaling and CD4⁺CD25⁺ T regulatory cells. Thus, CD80-dependent responses to myeloid suppressor cells may contribute to tumor tolerance and the progression of ovarian carcinoma.(Cancer Res 2006; 66(13): 6807-15)