We have shown that staurosporine (STSP) arrests normal human diploid fibroblasts in the G1 phase of the cell cycle at a time approximately 3 h after release from low-serum-induced G0 arrest. This initial temporal mapping of the STSP-induced restriction point was based on flow cytometric analyses that measured the onset of DNA synthesis after release from STSP and low-serum treatment. Here we show that the STSP-mediated arrest point distinctly differs from low-serum G0 arrest. We have found that cyclin D1 is expressed in STSP-arrested G1 fibroblasts but not in low-serum-arrested G0 fibroblasts, whereas cyclin-dependent kinase 4 (cdk4) and proliferating cell nuclear antigen (PCNA) are equivalently expressed under conditions of both STSP treatment and serum deprivation. Cdk4/cyclin D1/PCNA complexes are also formed in STSP-arrested G1 fibroblasts, but they are absent in serum-deprived G0 cells. The formation of cdk4/cyclin D1/PCNA complexes was found to coincide with the transcription and synthesis of cyclin D1, which indicates that the lack of available cyclin D1 is the limiting factor in cdk4/cyclin D1/PCNA complex formation in serum-deprived fibroblasts. This conclusion was further supported by the observation that cyclin D1-GST fusion protein binds cdk4 and PCNA when added to G0 cell extracts. Circumstantial evidence obtained in our studies and by other investigators suggests that STSP-induced arrest may be due to the inhibition of cdk-activating kinase.