Variant histone H3.3 is incorporated into nucleosomes by a mechanism that does not require DNA replication and has also been implicated as a potential mediator of epigenetic memory of active transcriptional states. In this study, we have used chromatin immunoprecipitation analysis to show that H3.3 is found mainly at the promoters of transcriptionally active genes. We also show that H3.3 combines with H3 acetylation and K4 methylation to form a stable mark that persists during mitosis. Our results suggest that H3.3 is deposited principally through the action of chromatin-remodelling complexes associated with transcriptional initiation, with deposition mediated by RNA polymerase II elongation having only a minor role.