Cyclin D1 gene overexpression is a frequent event in a number of human cancers. These observations have led to the suggestion that cyclin D1 alterations might play a role in the etiology of cancer. This possibility is supported by the finding that transfection of mammalian cells with cyclin D1 can accelerate progression through the G1 phase of the cell cycle. Moreover, cyclin D1 can function as an oncogene by cooperating with activated Ha-ras to transform primary rat embryo fibroblasts (REFs). In addition, cyclin D1 transgenics develop hyperplasia and neoplasia of the thymus and mammary gland. We have constructed a novel fusion gene consisting of full-length human cyclin D1 and cdk4 genes. This fusion gene was expressed in insect cells and the fusion protein was shown to be enzymatically active. The fusion gene was expressed in mammalian cells under the control of tet-repressor. This fusion gene immortalized primary REFs, and cooperated with activated Ha-ras to transform primary REFs, in terms of anchorage-independent growth in vitro and formation of tumors in vivo. Utilizing a tet-regulated gene expression system, we have shown that proliferation of stably transfected primary REFs in vitro and in vivo is dependent on the continued expression of the cyclin D1-cdk4 fusion gene. These cell lines could be useful in the discovery of novel cancer therapeutics to modulate cyclin D1.cdk4 activity.