Histone modifications and variants have key roles in the activation and silencing of genes. Phosphorylation of histone H3 at serine 10 and serine 28 is involved in transcriptional activation of genes responding to stress or mitogen-stimulated signaling pathways. The distribution of H3-modified isoforms in G0 phase chicken erythrocyte chromatin was investigated. H3 phosphorylated at serine 28 was found highly enriched in the active/competent gene fractions, as was H3 di- and trimethylated at lysine 4. The H3 variant H3.3 in this chromatin fraction was preferentially phosphorylated at serine 28. Conversely, H3 phosphorylated at serine 10 was present in all chromatin fractions, while H3 dimethylated at lysine 9 was associated with the chromatin-containing repressed genes. H3 phosphorylated at serine 28 was located at the promoter region of the transcriptionally active, but not competent, histone H5 and beta-globin genes. We provide evidence that H3.3 phosphorylated at serine 28 was present in labile nucleosomes. We propose that destabilized nucleosomes containing H3.3 phosphorylated at serine 28 aid in the dynamic disassembly-assembly of nucleosomes in active promoters.