We have immunolabeled human and mouse metaphase chromosomes with antibodies specific for the acetylated isoforms of histone H4. All chromosomes were labeled in regions corresponding to conventional R bands (regions enriched in coding DNA), except for a single chromosome in female cells, which was largely unlabeled and which we have identified as the inactive X (Xl). Three sharply defined lmmunofluorescent bands, enhanced by butyrate pretreatment, were observed in homologous positions on the human and mouse Xi, showing limited, regional persistence of H4 acetylation. Two of these bands are in cytogenetic regions known to contain genes expressed on Xi. We propose that H4 hyperacetylation defines regions of the genome containing potentially transcriptionally active chromatin, while virtual absence of H4 acetylation defines both constitutlve and facultative heterochromatin.