Nucleosomes are the fundamental units of chromatin, and the process of nucleosome modification is thought to maintain epigenetic chromatin states. Histone variants provide another means of distinguishing chromatin states, and three different H3 histories are found in most eukaryotes. We have used a Drosophila cell line system to investigate the dynamics of H3 histone assembly into chromatin. Conventional H3 is produced in abundance at S phase and is assembled into chromatin in a strictly replication-dependent manner. Drosophila centromeric histone, Cid, assembles via a replication-independent (RI) deposition pathway, where Cid production appears to be coordinated with centromere replication in mid-S phase. Late-replicating pericentric heterochromatin may help to maintain embedded centromeres by blocking conventional nucleosome assembly earlier in S phase, thereby favoring the deposition of centromeric nucleosomes. The third H3 histone, H3.3, has received little recent attention, as it differs from H3 at only four amino acid positions. We have discovered a novel mechanism of chromatin regulation whereby H3.3 is deposited at particular loci, including active rDNA arrays. Single-amino acid changes of H3 towards H3.3 allow RI deposition, indicating that RI is the default pathway for chromatin assembly. In contrast to replication-coupled assembly, RI deposition does not require the N-terminal tail. H3.3 is the exclusive substrate for RI deposition, and its counterpart is the only substrate retained in yeast. RI substitution of H3.3 provides a mechanism for the immediate activation of genes that are silenced by histone modification. Inheritance of newly deposited nucleosomes may then mark sites as active loci.