The c-ski proto-oncogene product (c-Ski) acts as a co-repressor and binds to other co-repressors N-CoR/SMRT and mSin3A which form a complex with histone deacetylase (HDAC). c-Ski mediates the transcriptional repression by a number of repressors, including nuclear hormone receptors and Mad. c-Ski also directly binds to, and recruits the HDAC complex to Smads, leading to inhibition of tumor growth factor-beta (TGF-beta) signaling. This is consistent with the function of ski as an oncogene. Here we show that loss of one copy of c-ski increases susceptibility to tumorigenesis in mice. When challenged with a chemical carcinogen, c-ski heterozygous mice showed an increased level of tumor formation relative to wild-type mice. In addition, c-ski-deficient mouse embryonic fibroblasts (MEFs) had increased proliferative capacity, whereas overexpression of c-Ski suppressed the proliferation. Furthermore, the introduction of activated Ki-ras into c-ski-deficient MEFs resulted in neoplastic transformation. These findings demonstrate that c-ski acts as a tumor suppressor in some types of cells. The level of cdc25A mRNA, which is down regulated by two tumor suppressor gene products, Rb and Mad, was upregulated in c-ski-deficient MEFs, whereas it decreased by overexpressing c-Ski in MEFs. This is consistent with the fact that c-Ski acts as a co-repressor of Mad and Rb. These results support the view that the decreased activities of Mad and Rb in ski-deficient cells at least partly contribute to enhanced proliferation and susceptibility to tumorigenesis. Human c-ski gene was mapped to a region close to the p73 tumor suppressor gene at the 1p36.3 locus, which is already known to contain multiple uncharacterized tumor suppressor genes.