p21cip1/waf1/sdi1 is a universal cyclin-Cdk kinase inhibitor that has two functional domains; one binds and inhibits cyclin-Cdk activity and the other binds PCNA and thereby inhibits elongation by DNA polymerase. When transiently expressed in hamster BHK21 cells we found that human p21 was able to cause cell cycle arrest in G1 phase; this arrest was counteracted by coexpression of E2F-1 or SV40 large T antigen. To study the effect of p21 overexpression in vivo, BHK21 cell clones inducibly expressing human p21 (Tet-p21) driven by the tetracycline (Tet)-repressible promoter were established. The maximum induced p21 levels in the absence of Tet were estimated to be ten times that of endogenous hamster p21. As p21 levels rose following removal of Tet, p21-associated histone H1 kinase activity was increased and concomitantly cell growth and DNA synthesis were reduced. Tet-p21 BHK21 cells became arrested in G1 phase and lost colony forming ability irreversibly 2-4 days after removal of Tet. The induction of cyclin E- and cyclin A-associated kinase activities was diminished when G0-synchronized Tet-p21 BHK21 cells were serum stimulated in the absence of Tet. Increased binding of p21 to PCNA and cyclin D1-Cdk4 was detected in induced cells. Overexpression of p21 led to cell death in BHK21 cells at 39.5 degrees C within 4 days.