Mcl-1 is an antiapoptotic member of the Bcl-2 family that plays an important role in cell survival. We demonstrate that proteasome-dependent regulation of Mcl-1 plays a critical role in renal tubular epithelial cell injury from cisplatin. Protein levels of Mcl-1 rapidly declined in a time-dependent manner following cisplatin treatment of LLC-PK(1) cells. However, mRNA levels of Mcl-1 were not altered following cisplatin treatment. Expression of other antiapoptotic members of the Bcl-2 family such as Bcl-2 and BclxL was not affected by cisplatin treatment. Cisplatin-induced loss of Mcl-1 occurs at the same time as the mitochondrial release of cytochrome c, activation of caspase-3, and initiation of apoptosis. Treatment of cells with cycloheximide, a protein synthesis inhibitor, revealed rapid turnover of Mcl-1. In addition, treatment with cycloheximide in the presence or absence of cisplatin demonstrated that cisplatin-induced loss of Mcl-1 results from posttranslational degradation rather than transcriptional inhibition. Overexpression of Mcl-1 protected cells from cisplatin-induced caspase-3 activation and apoptosis. Preincubating cells with the proteasome inhibitor MG-132 or lactacystin not only restored cisplatin-induced loss of Mcl-1 but also resulted in an accumulation of Mcl-1 that exceeded basal levels; however, Bcl-2 and BclxL levels did not change in response to MG-132 or lactacystin. The proteasome inhibitors effectively blocked cisplatin-induced mitochondrial release of cytochrome c, caspase-3 activation, and apoptosis. These studies suggest that proteasome regulation of Mcl-1 is crucial in the cisplatin-induced apoptosis via the mitochondrial apoptotic pathway and that Mcl-1 is an important therapeutic target in cisplatin injury to renal tubular epithelial cells.