In the following report the relationship between histone methylation and histone acetylation has been examined in HeLa cells to better define the distribution of these two modifications. By labeling methylated histones in the presence or absence of sodium butyrate, we have found that the methylation of H3 is much more targeted to rapidly acetylated chromatin than is the methylation of H4, which largely involves the unacetylated subtype even in the presence of butyrate. Newly methylated H3 is highly likely to be complexed in nucleosomes that contain acetylated H4, as determined by immunoprecipitating radiolabeled chromatin with antibodies specific for acetylated H4 isoforms. In contrast, dynamically methylated H4 is underrepresented in acetylated chromatin, relative to newly methylated H3. The preferential methylation of acetylated H3 continues after pretreatment of cells with cycloheximide, indicating that not all acetylation-related methylation is associated with histone synthesis. This was confirmed by analyzing histone methylation in cells arrested at the G1/S boundary, in which histone synthesis was sharply lowered (relative to randomly cycling cells): under these conditions H3 methylation declined only approximately 4-fold, although ongoing methylation of H4 decreased approximately 20-fold. The continuing methylation of H3 in arrested cells included all H3 sequence variants, was selective for acetylated H3, and coincided with methyl group turnover that could not be ascribed to histone replacement synthesis. Most newly methylated H3 in arrested cells was complexed with acetylated H4 in chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)