The chromosomal fragments produced by nuclease digestion of freshly replicated chromatin migrate more rapidly relative to bulk chromatin when analyzed in nucleoprotein gels. The cause of the anomalous migration has been studied and the evidence indicates that rather than reflecting a shorter nucleosomal repeat in vivo that it may be a consequence of nucleosome sliding during the digestion itself. The distinct electrophoretic characteristics of nucleosomal material containing newly replicated DNA have enabled us to examine their histone composition by two dimensional electrophoresis. We find that nucleosomes containing new DNA also contain newly synthesized histones H3 and H4. In contrast more than 50% of newly synthesized H2A and H2B, and essentially all of new H1, are deposited at sites on the bulk chromatin distinct from that material containing newly replicated DNA. In addition we show that newly synthesized histones H3 and H4 are bound unusually weakly when they first become associated with the chromatin.