Apaf-1 and the cysteine proteases known as caspases are genes central to the intrinsic apoptotic pathway in the retina. Previously, we have shown that histone deacetylase (HDAC) activity regulates Apaf-1 expression in the retina. In this study, we unravel the detailed molecular mechanism of HDAC-mediated regulation of Apaf-1 initially by use of a cell line (661W), which expresses some cone-specific genes and then by means of an ex vivo retinal explant system. Inhibition of HDAC activity by trichostatin A (TSA) up-regulates Apaf-1 expression, which precedes the induction of apoptosis. Furthermore, by a bioinformatics approach, we identify E2F-1 and p53 binding sites on the mouse Apaf-1 promoter and show by chromatin immunoprecipitation assays that these sites are occupied in vitro and that treatment with TSA results in increased binding of E2F-1 and p53 to the Apaf-1 promoter. By performing siRNA to these transcription factors, we illustrate that they govern Apaf-1 expression levels in vitro. Finally, in a retinal explant system, we show that similar to our 661W results, E2F-1 and p53 are up-regulated after inhibition of HDAC activity in the retina. This correlates with our previous observation in the explant system that Apaf-1 expression increases significantly and leads to an induction of apoptosis after inhibition of HDAC activity. Overall, we propose a role for HDAC activity, E2F-1, and p53 in the regulation of Apaf-1 expression in 661W cells; initial data also indicate a regulatory role in the retina.