p53, the most commonly mutated gene in cancer cells, directs cell cycle arrest or induces programmed cell death (apoptosis) in response to stress. It has been demonstrated that p53 activity is up-regulated in part by posttranslational acetylation. In agreement with these observations, here we show that mammalian histone deacetylase (HDAC)-1, -2, and -3 are all capable of downregulating p53 function. Down-regulation of p53 activity by HDACs is HDAC dosage-dependent, requires the deacetylase activity of HDACs, and depends on the region of p53 that is acetylated by p300/CREB-binding protein (CBP). These results suggest that interactions of p53 and HDACs likely result in p53 deacetylation, thereby reducing its transcriptional activity. In support of this idea, GST pull-down and immunoprecipitation assays show that p53 interacts with HDAC1 both in vitro and in vivo. Furthermore, a pre-acetylated p53 peptide was significantly deacetylated by immunoprecipitated wild type HDAC1 but not deacetylase mutant. Also, coexpression of HDAC1 greatly reduced the in vivo acetylation level of p53. Finally, we report that the activation potential of p53 on the BAX promoter, a natural p53-responsive system, is reduced in the presence of HDACs. Taken together, our findings indicate that deacetylation of p53 by histone deacetylases is likely to be part of the mechanisms that control the physiological activity of p53.