In vitro DNA synthesis was studied in HeLa cell nuclei, with emphasis on the question of whether newly replicated DNA is associated with nucleosomes. The newly replicated DNA was twice as sensitive to digestion by micrococcal nuclease as mature chromatin DNA, reaching a limit digest at 20-25% acid-insoluble product. Examination of the intermediates of digestion by micrococcal nuclease showed the nuclease-resistant, new DNA to be complexed in nucleosomes. However, structural differences were evident at both the polynucleosomal and the core particle level. The nucleosomes on newly replicated DNA were arranged with a repeat size of 165-170 base pairs-i.e., smaller than the 185-base-pair repeat of mature chromatin. The heterogeneity of polynucleosomal multimers, evident in digests of whole chromatin, was reduced in newly replicated chromatin such that the multimers resolved as sharply defined bands. Nucleosomal core particles associated with newly replicated DNA had a different conformation from particles in mature chromatin based on the following lines of evidence: (i) during micrococcal nuclease digestion, the monomer nucleosomes did not accumulate but were rapidly degraded under certain conditions; (ii) micrococcal nuclease limit digest patterns and DNase I digestion patterns, both of which reflect internal nucleosomal protein DNA associations, differed significantly from control patterns. These findings bear directly on models postulated for nucleosome-DNA interactions during chromation replication. A possible mechanism to account for the conformational change and its role in replication are discussed.