Sam68 is a multimeric 68-kDa RNA-binding nuclear protein of unknown function that interacts with, and is tyrosine-phosphorylated by, the oncogenic protein Src during mitosis. Random homozygous knock-out (RHKO) is a retroviral-based antisense RNA strategy that can identify chromosomal genes whose functional disablement leads to reversible tumorigenic capabilities. Here we report that RHKO-induced Sam68 deficiency results in neoplastic transformation of murine NIH3T3 fibroblasts. Whereas simple haploinsufficiency of Sam68 produced by insertion mutagenesis in a single chromosomal allele did not detectably affect cell growth, reduction of Sam68 protein to <25% of the wild type level was associated with anchorage-independent growth, defective contact inhibition, and the ability to form metastatic tumors in nude mice. These properties were reversed by cessation of RHKO inactivation. Our findings, which indicate that the Sam68 protein level can prominently affect cell proliferation, implicate Sam68 function in tumorigenesis. Consistent with these results is evidence that cells undergoing mitosis show a dramatic reduction in the level of Sam68 protein.