Chromatin can be differentiated by the deposition of variant histones at centromeres, active genes, and silent loci. Variant histones are assembled into nucleosomes in a replication-independent manner, in contrast to assembly of bulk chromatin that is coupled to replication. Recent in vitro studies have provided the first glimpses of protein machines dedicated to building and replacing alternative nucleosomes. They deposit variant H2A and H3 histones and are targeted to particular functional sites in the genome. Differences between variant and canonical histones can have profound consequences, either for delivery of the histones to sites of assembly or for their function after incorporation into chromatin. Recent studies have also revealed connections between assembly of variant nucleosomes, chromatin remodeling, and histone post-translational modification. Taken together, these findings indicate that chromosome architecture can be highly dynamic at the most fundamental level, with epigenetic consequences.