Histone H4 isoforms acetylated at lysines 5, 8, 12, or 16 have been shown, by indirect immunofluorescence with site-specific antisera, to have distinct patterns of distribution in interphase, polytene chromosomes from Drosophila larvae. H4 molecules acetylated at lysines 5 or 8 are distributed in overlapping, but nonidentical, islands throughout the euchromatic chromosome arms. f% Heterochromatin in the chromocenter is depleted in these isoforms, but relatively enriched in H4 acetylated at lysine 12. H4 acetylated at lysine 16 is found at numerous sites along the transcriptionally hyperactive X chromosome in male larvae, but not in male autosomes or any chromosome in female cells. These findings support the hypothesis that H4 molecules acetylated at particular sites mediate unique and specific effects on chromatin function.