Complex organisms are able to rapidly induce select genes among thousands in response to diverse environmental cues. This occurs in the context of large genomes condensed with histone proteins into chromatin. The macrophage response to pathogen sensing, for example, rapidly engages highly conserved signaling pathways and transcription factors (TFs) for coordination of inflammatory gene induction1–3. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a feature of inflammatory genes and, in general, dynamically regulated chromatin and transcription4–7. However, little is known of how chromatin is regulated at rapidly induced genes and what features of H3.3, conserved from yeast to human, might enable rapid and high-level transcription. The amino-terminus of H3.3 contains a unique serine residue as compared with alanine residues found in “canonical” H3.1/2. We find that this H3.3-specific serine residue, H3.3S31, is phosphorylated (H3.3S31ph) in a stimulation-dependent manner along the gene bodies of rapidly induced response genes in mouse macrophages responding to pathogen sensing. Further, this selective mark of stimulation-responsive genes directly engages histone methyltransferase (HMT) SETD2, a component of the active transcription machinery. Our structure-function studies reveal that a conserved positively charged cleft in SETD2 contacts H3.3S31ph and specifies preferential methylation of H3.3S31ph nucleosomes. We propose that features of H3.3 at stimulation induced genes, including H3.3S31ph, afford preferential access to the transcription apparatus. Our results provide insight into the function of ancestral histone variant H3.3 and the dedicated epigenetic mechanisms that enable rapid gene induction, with implications for understanding and treating inflammation. A poorly understood feature of stimulation-induced genes is their ability to effectively engage the general transcription machinery for rapid expression. Selective, induced gene transcription, for example during heat shock8 or the inflammatory response, occurs rapidly and robustly, despite these genes’ de novo expression among thousands of constitutively expressed genes. We considered that stimulation-induced transcription may be controlled by dedicated epigenetic mechanisms in cooperation with signal-activated transcription factors (TFs). Among stimulation-responsive features of chromatin, histone phosphorylation can be an efficient and potent means of transmitting signals via kinase cascades to chromatin regions associated with stimulation-responsive genes with the potential to augment their transcription9–14. H3.3 is the conserved, ancestral H3 variant and the only H3 present in some simple eukaryotes, including S. cerevisiae. In complex organisms, H3.3 is uniquely expressed outside of the cell cycle and plays a variety of roles in transcription, genomic stability and mitosis, while so-called “canonical” H3.1/2 histones are expressed in a “replication-dependent” manner and provide a principal packaging role to accommodate the doubling genome15,16. The amino-terminal H3.3 ‘tail’ differs from that of H3.1/2 by a single amino acid, a serine at position 31 in H3.3 in place of an alanine in H3.1/2 (Fig. 1A and fig. S1A). Despite the well-characterized enrichment of H3.3 in dynamic chromatin, the potential regulatory roles of H3.3S31 and H3.3-specific phosphorylation are unknown4–7,17. Here, we report that H3.3 phosphorylation at the conserved and H3.3-specific serine 31 (H3.3S31ph) amplifies the rapid, high-level transcription of stimulation-induced gene expression. We present a specific biophysical mechanism that provides these select genes with augmented transcriptional capacity.