Mullerian inhibiting substance (MIS) inhibits breast cancer cell growthin vitro. To extend the use of MIS to treat breast cancer, it is essential to test the responsiveness of mammary tumor growth to MISin vivo. Mammary tumors arising in the C3(1) T antigen mouse model expressed the MIS type II receptor, and MISin vitroinhibited the growth of cells derived from tumors. Administration of MIS to mice was associated with a lower number of palpable mammary tumors compared with vehicle-treated mice (P= 0.048), and the mean mammary tumor weight in the MIS-treated group was significantly lower compared with the control group (P= 0.029). Analysis of proliferating cell nuclear antigen (PCNA) expression and caspase-3 cleavage in tumors revealed that exposure to MIS was associated with decreased proliferation and increased apoptosis, respectively, and was not caused by a decline in T antigen expression. The effect of MIS on tumor growth was also evaluated on xenografted human breast cancer cell line MDA-MB-468, which is estrogen receptor- and retinoblastoma-negative and expresses mutant p53, and thus complements the C3(1)Tag mouse mammary tumors that do not express estrogen receptor and have functional inactivation of retinoblastoma and p53. In agreement with results observed in the transgenic mice, MIS decreased the rate of MDA-MB-468 tumor growth and the gain in mean tumor volume in severe combined immunodeficient mice compared with vehicle-treated controls (P= 0.004). These results suggest that MIS can suppress the growth of mammary tumorsin vivo.