DNA in eukaryotic cells is packaged into nucleosomes, the structural unit of chromatin. Both DNA and bulk histones are extremely long-lived, because old DNA strands and histones are retained when chromatin duplicates. In contrast, we find that the Drosophila HSP70 genes rapidly lose histone H3 and acquire variant H3.3 histones as they are induced. Histone replacement does not occur at artificial HSP70 promoter arrays, demonstrating that transcription is required for H3.3 deposition. The H3.3 histone is enriched in all active chromatin and throughout large transcription units, implying that deposition occurs during transcription elongation. Strikingly, we observed that the stability of chromatin-bound H3.3 differs between loci: H3.3 turns over at continually active rDNA genes, but becomes stable at induced HSP70 genes that have shut down. We conclude that H3.3 deposition is coupled to transcription, and continues while a gene is active. Repeated histone replacement suggests a mechanism to both maintain the structure of chromatin and access to DNA at active genes.