Cyclin-dependent kinase 1 (CDK1) is a major component of the cell cycle progression engine. Recently, several investigations provided evidence demonstrating that unscheduled CDK1 activation may also be involved in apoptosis in cancerous cells. In this article, we demonstrate that X-ray irradiation induced G1 arrest in MOLT-4 lymphocytic leukemia cells, the arrest being accompanied by reduction in the activity of CDK2, but increased CDK1 activity and cell apoptosis in the G1 phase. Interestingly, this increase in CDK1 and apoptosis by ionizing radiation was prevented by pretreatment with the CDK1 inhibitor, roscovitine, suggesting that CDK1 kinase activity is required for radiation-induced apoptotic cell death in this model system. Furthermore, cyclin B1 and CDK1 were detected co-localizing and associating in G1 phase MOLT-4 cells, with the cellular lysates from these cells revealing a genotoxic stress-induced increase in CDK1 phosphorylation (Thr-161) and dephosphorylation (Tyr-15), as analyzed by postsorting immunoprecipitation and immunoblotting. Finally, X-irradiation was found to increase Bcl-2 phosphorylation in G1 phase cells. Taken together, these novel findings suggest that CDK1 is activated by unscheduled accumulation of cyclin B1 in G1 phase cells exposed to X-ray, and that CDK1 activation, at the wrong time and in the wrong phase, may directly or indirectly trigger a Bcl-2-dependent signaling pathway leading to apoptotic cell death in MOLT-4 cells.