During nucleosome assembly in vivo, newly synthesized histone H4 is specifically diacetylated on lysines 5 and 12 within the H4 NH(2)-terminal tail domain. The highly conserved "K5/K12" deposition pattern of acetylation is thought to be generated by the Hat1 histone acetyltransferase, which in vivo is found in the HAT-B complex. In the following report, the activity and substrate specificity of the human HAT-B complex and of recombinant yeast Hat1p have been examined, using synthetic H4 NH(2)-terminal peptides as substrates. As expected, the unacetylated H4 peptide was a good substrate for acetylation by yeast Hat1p and human HAT-B, while the K5/K12-diacetylated peptide was not significantly acetylated. Notably, an H4 peptide previously diacetylated on lysines 8 and 16 was a very poor substrate for acetylation by either yeast Hat1p or human HAT-B. Treating the K8/K16-diacetylated peptide with histone deacetylase prior to the HAT-B reaction raised acetylation at K5/K12 to 70-80% of control levels. These results present strong support for the model of H4-Hat1p interaction proposed by Dutnall et al. (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998) Cell 94, 427-438) and provide evidence for the first time that site-specific acetylation of histones can regulate the acetylation of other substrate sites.