In eukaryotic cells, transcriptionally active chromatin and inactive chromatin exist in dissimilar configurations which correlate with their functional states. The present study asks whether the differences in structure and function of active and inactive chromatin are also reflected in the process of replication. We have used mild micrococcal nuclease digestion [Bloom, K.S., & Anderson, J.N. (1978) Cell (Cambridge, Mass.) 15, 141-150] to release selectively an active chromatin fraction, S1, from MSB cell nuclei. The S1 fraction is greater than 6-fold enriched in sequences complementary to polyadenylated RNA and thus consists of virtually pure active chromatin. As isolated, the S1 chromatin comprises mononucleosomes which contain approximately 160 base pairs of DNA bound by core histones but which are enriched in non-histones and free of histone H1. Chemical cross-linking of S1 chromatin yields octamers containing the normal stoichiometry of core histones. When MSB cells are pulse labeled with isotopically dense amino acids and the recovered S1 chromatin is isolated and cross-linked, the labeled histone octamers are found in a single hybrid density octamer band on isopycnic gradients. These results indicate that, in contrast to the conservative assembly of transcriptionally inactive nucleosomes, the deposition of histones during the replication of active chromatin follows a nonrandom, semiconservative pathway.