Alternative use of genes of the closely-related pp32 family is a common occurrence in human prostate cancer. pp32r1 and pp32r2, the oncogenic members of the pp32 family, are expressed in prostatic adenocarcinoma, while adjacent benign prostate continues to express pp32. This study focuses upon the role of pp32 in tumor suppression. We demonstrate that antisense inhibition of pp32 in NIH3T3 cells leads to a variety of phenotypic changes associated with transformation including reduced serum dependence and loss of contact inhibition. NIH3T3 cells with antisense-inhibited pp32 are not tumorigenic, but are markedly more susceptible to oncogenic stimuli such as ras. In contrast, constitutive expression of pp32 abolishes ras mediated transformation in vitro and tumorigenesis in vivo. These data demonstrate, from the functional aspect, that pp32 acts as a tumor suppressor. Furthermore, inactivation of pp32 function through alternative gene use may be a critical event in tumor evolution and progression.