p53 regulates transcription of one anti-apoptotic and four pro-apoptotic members of the BCL-2 family, but nothing is known about the regulation of MCL-1, another antiapoptotic member of this family, by p53. Confocal microscopic analysis of COS1, HEK 293 and HeLa cells transfected with a p53 expression plasmid demonstrated a decrease in the signal of endogenous MCL-1 compared to neighboring non-transfected cells. Transcription regulation assays showed that the 1826 bp human MCL-1 promoter fragment was repressed up to 30-fold by wild-type p53 in a dose-dependent manner. As shown by electrophoretic mobility shift assays, Sp1 binding to the sites located in the -295 to +16 MCL-1 promoter fragment was decreased in the presence of p53. However, the MCL-1 promoter devoid of all Sp1 binding sites was still repressed by p53, albeit 2-fold weaker than the wild-type promoter. Overexpression of Sp1 reduced p53-dependent repression of the MCL-1 promoter only up to 2.2-fold. Transcription regulation assays performed with MCL-1 promoter deletion mutants showed that most of the p53 inhibitory effect was mediated by the -41 to +16 bp promoter fragment containing binding sites only for TATA-binding protein and other basal transcription factors. We propose a novel, promoter-based mechanism by which p53 down-regulates expression of the antiapoptotic MCL-1 protein.