THE yeast transcriptional adaptor(1-3), Gcn5p, is a catalytic subunit of a nuclear (type A) histone acetyltransferase linking histone acetylation to gene activation(4-6). Here we report that Gcn5p acetylates histones H3 and H4 non-randomly at specific lysines in the amino-terminal domains. Lysine 14 of H3 and lysines 8 and 16 of H4 are highly preferred acetylation sites for Gcn5p. We also demonstrate that lysine 9 is the preferred position of acetylation in newly synthesized yeast H3 in vivo. This finding, along with the fact that lysines 5 and 12 in H4 are predominant acetylation sites during chromatin assembly of many organisms(7-11), indicates that Gcn5p acetylates a distinct set of lysines that do not overlap with those sites characteristically used by type B histone acetyltransferases for histone deposition and chromatin assembly.