Electrophoresis, Western blotting and immunostaining with antibodies specific for histone H4 acetylated at lysines 5, 8, 12, or 16, were used to define patterns of H4 acetylation in cell lines from humans (HL60) and the fruit fly Drosophila (S2, Kc). In human cells, the mono-acetylated isoform H4Ac 1 is acetylated predominantly at just one of the four possible lysine residues, lysine 16. This is the first step in the progressive acetylation of H4. In contrast, in Drosophila, H4Ac 1 is acetylated at lysines 5, 8, or 12 with approximately equal frequency. Fundamental differences appear to exist in control of H4 acetylation in different species, despite the evolutionary conservation of acetylation sites.