During periods of active DNA replication and chromatin assembly, newly synthesized histone H4 is deposited in a diacetylated form. In Tetrahymena, a specific pair of residues, lysines 4 and 11, has been shown to undergo this modification in vivo (Chicoine, L. G., Schulman, I. G., Richman, R., Cook, R. G., and Allis, C. D. (1986) J. Biol. Chem. 261, 1071-1076). Presumably, this reaction is catalyzed, at least in part, by histone acetyltransferases (HAT) of the B type, cytoplasmic enzymes displaying strong preference for free, non-chromatin-bound H4. To investigate which lysines are preferred acetylation sites in H4 from other organisms, a cytoplasmic HAT B activity was prepared from Drosophila embryos and used to acetylate H4 from several species. When H4 or synthetic, NH2-terminal peptides from Tetrahymena were used as unblocked substrates, direct microsequence analyses showed that [3H]acetate was preferentially incorporated at lysine 11 with little, if any, incorporation at other conserved, acetylatable lysines. Drosophila H4 was chemically deblocked following its acetylation in vitro using conditions that do not deacetylate internal lysines. Direct sequence analysis verified the correct NH2-terminal sequence of Drosophila H4 and demonstrated that [3H]acetate incorporation occurred preferentially on lysine 12, the residue analogous to lysine 11 in Tetrahymena. These data show remarkable preference for lysine 11/12 by the Drosophila HAT B activity in vitro and provide support for the assertion that this activity functions to acetylate new H4, at least in part, for deposition and chromatin assembly in vivo. Since most H4s, like Drosophila, are blocked at their amino termini by an acetylthreonine or acetylserine, our results demonstrate that this deblocking and microsequencing strategy can be used to study acetylation site utilization in H4 and presumably other core histones NH2 terminally blocked with these residues.