The histone acetyltransferases p300 and cAMP-responsive element-binding protein-binding protein (CBP) are required for the execution of critical biological functions such as proliferation, differentiation, and apoptosis. Both proteins are believed to regulate the activity of a large number of general and cell-specific transcription factors. Here we demonstrate a dramatic decrease in the total cellular levels of p300 and CBP with increasing population doublings of human normal melanocytes. We show that one consequence of p300 depletion is transcriptional down-regulation of the cyclin E gene, caused by deacetylation of histones at its promoter. The cyclin E promoter was activated by p300 and the histone deacetylase inhibitor trichostatin A. Conversely, the cyclin E promoter was repressed by wild-type Retinoblastoma tumor suppressor p105 protein (pRB) and by a dominant negative p300 mutant (DN p300) that lacks histone acetyltransferase activity. We also provide evidence of the alternative recruitment of p300 and histone deacetylase 1 to the cyclin E promoter in proliferating and senescent melanocytes, respectively. The biological significance of these results was established by showing that block of p300 activity by overexpression of DN p300 or by Lys-CoA, a specific chemical inhibitor of p300, resulted in growth inhibition, down-regulation of cyclin E, and activation of the senescence-associated beta-galactosidase marker in human melanocytes and melanoma cells. Together, these results provide evidence for the essential role of p300 in the regulation of proliferation and senescence in cells from melanocytic origin.