We initiated studies to investigate the involvement of the murine retinoblastoma (RB) gene in mammary carcinogenesis using cell lines derived from mammary glands of irradiated mice. We found that the RB mRNA levels as well as the amounts of the nuclear phosphoprotein were significantly reduced as the cells progressed in vitro from non-tumorigenic to tumorigenic stages. To further investigate RB gene expression with cellular development and tumorigenicity, we transfected malignant cells with expression vectors containing the murine RB cDNA driven by either the SV40 or the mouse metallothionein promoter sequences. The neomycin resistant gene was included in both vectors and was used as a selective marker for the transfected cells. Cells with reduced levels of endogenous RB mRNA were stably transfected and showed increased expression of RB. In addition, the morphology of these cells were altered and their growth rates in culture were reduced. Injection of the transfected cells into host mice resulted in a delayed onset of tumors compared with nontransfected parental cells. Our studies provide experimental data to confirm that loss of RB gene activity is involved in neoplastic transformation of cells and support the multistep theory of carcinogenesis.