The N-terminal domains of all four core histones are subject to reversible acetylation at certain lysine residues. This modification has been functionally linked to transcription, histone deposition at replication and to histone removal during spermatogenesis. To increase understanding of the significance of this modification we have studied the specificity of site utilisation in the monoacetyl, diacetyl and triacetyl forms of histones H3, H4 and H2B (histone H2A has only a single modification site), using pig thymus and HeLa cells as the source of histones. The HeLa histones were extracted from cells grown both with and without butyrate treatment. It is found that for histone H3 there is a fairly strict order of site occupancy: Lys14, followed by Lys23, followed by Lys18 in both pig and HeLa histones. Since the order and specificity is the same when butyrate is added to the HeLa cell cultures, we conclude that addition of the fatty acid does not scramble the specificity of site utilisation, but merely allows more of the natural forms of modified histone to accumulate. For histone H4, the monoacetyl form is exclusively modified at Lys16, but further addition of acetyl groups is less specific and progresses through sites 12, 8 and 5 in an N-terminal direction. Similar results were obtained for H4 from both pig thymus and butyrate-treated HeLa cells. Histone H2B could be studied in detail only from butyrate-treated HeLa cells and a much lower level of site specificity was found: sites 12 and 15 were preferred to the more N- and C-terminal sites at Lys5 and Lys20. The data reinforces the view that lysine acetylation in core histones is a very specific phenomenon that plays several functionally distinct roles. The high degree of site specificity makes it unlikely that the structural effects of acetylation are mediated merely by a generalised reduction of charge in the histone N-terminal domains.