Protein complexes containing cyclins and cyclin-dependent protein kinases (cdks) have been shown to be rearranged in both spontaneous and viral tumor antigen-transformed cells. We have examined G1- and S-phase cyclin/cdk complexes as a function of the neoplastic progression of human diploid fibroblasts transfected with the SV40 large T antigen. We find that the expression of cyclin D1 and its association with proliferating cell nuclear antigen (PCNA) and Waf1 remain unchanged in precrisis human fibroblasts transfected with SV40 large T antigen. However, in these same cells the association of cdk4 with cyclin D1, PCNA, and Waf1 is disrupted. Upon immortalization, cyclin D1 protein expression is decreased, and binding of both PCNA and Waf1 with the remaining cyclin D1 is reduced. In contrast, large T antigen increased the expression of cyclin A and cyclin E proteins in both precrisis and immortal cells and did not reduce the binding of PCNA or Waf1 to either cdk2 or cyclin A proteins. These results show that large T-antigen expression in human fibroblasts selectively uncouples cyclin D1 from cdk4, and subsequent immortalization of these cells results in additional changes to the cyclin D1-dependent cell cycle regulatory pathways.