The p16 gene is a candidate tumor suppressor, because mutation of the gene has been reported in many transformed cell lines and some primary tumor tissues. We have examined this possibility in murine cell lines (NIH3T3 and RSV-M) which lack p16 gene expression. Full-length human p16 cDNA was obtained from a HeLa cell line using polymerase chain reaction amplification. We constructed two separate retrovirus vectors carrying this p16 cDNA. First, we transduced the p16 cDNA into the murine cell lines using a retrovirus vector harboring the neomycin-resistance gene. The p16 gene-transduced cells formed no colonies after selection with G418, in contrast to the vector-transduced cells. Next, we used another retrovirus vector that expresses both the p16 cDNA and the Lac Z gene, which enabled us to distinguish affected cells from unaffected ones. Proliferation of the p16 gene-transduced cells was markedly inhibited and morphological change in the cells was also observed. Thus, we concluded that the p16 gene has an antiproliferative effect on the cell cycle and that the loss of its function may play a major role in dysregulated proliferation of the cells.