The nature of histone acetylation events in active chromatin is an important issue in transcriptional regulation. We have systematically analyzed the ability of p300, either alone or in response to an interacting activator, to acetylate specific recombinant histones in the context of free histones, histone octamers, or nucleosomal arrays. Our results indicate that p300 has an intrinsic ability to acetylate all core histones but that the level and specificity of histone acetylation is indeed context-dependent. Thus, H3 and H4 are preferentially acetylated in free octamers, whereas all histones are nearly equally acetylated, in an activator-dependent manner, in chromatin. Moreover, H3 and H4 show H2A and H2B tail-independent acetylation in chromatin, whereas maximal H2A and H2B acetylation in this context is dependent upon H3 and H4 tails (but not their acetylation). In further support of an apparent intrinsic preference of p300 for the H3 and H4 tails, as well as an important role for direct interactions of p300 with unacetylated H3 and H4 tails in both acetylation and transcription, we have shown that p300 selectively acetylates isolated H3 and H4 tails, that p300 strongly and selectively binds to free unacetylated H3 and H4 tails, and that p300-mediated acetylation of nucleosomal histones and transcriptional activation are selectively inhibited by isolated (unacetylated) H3 and H4 tails.