Ultraviolet (UV) irradiation of HeLa cells triggers an apoptotic response mediated by mitochondria. Biochemical analysis of this response revealed that the elimination of cytosolic inhibitors is required for mitochondrial release of cytochrome c and subsequent caspase activation. These inhibitors were found to be Mcl-1 and Bcl-x_L, two antiapoptotic members of the Bcl-2 family. Following UV treatment, Mcl-1 protein synthesis is blocked, the existing pool of Mcl-1 protein is rapidly degraded by the proteasome, and cytosolic Bcl-x_L translocates to the mitochondria. These events are sequential; the elimination of Mcl-1 is required for the translocation of Bcl-x_L. The disappearance of Mcl-1 is also required for other mitochondrial apoptotic events including Bax translocation, cytochrome c release, and caspase activation.