Phasing of nucleosomes on SV40 DNA was studied by the reconstitution of chromatin from SV40 DNA form I and core histones. The reconstituted chromatin sedimented with increasing S values as the histone : DNA ratios increased, and the buoyant densities in CsCl decreased concomitantly. The average repeat lengths of nucleosomes in the chromatin reconstituted at ratios of 1.0 and 1.5 were estimated to be 168 and 143 base pairs, respectively, by electrophoretic analysis of DNA fragments generated by micrococcal nuclease digestion. The chromatin generated a series of DNA bands that differed in size by about 10 nucleotides upon DNase I digestion followed by heat-denaturation. Phasing of nucleosomes was probed by the use of single-site restriction endonucleases, EcoRI, BamH1, BglI, and HpaII: the latter two cleave DNA at and near the origin of DNA replication and transcription. The form I DNA in the chromatin reconstituted at ratios of 0.5, 1.0, and 1.5 was cleaved up to 60 to 80%, 20 to 60%, and 0 to 10%, respectively. Although the frequency of cleavage by these enzymes was not very different at the ratio 0.5, the BglI site became relatively more susceptible than the other sites at the ratio 1.0. At the ratio 1.5, the DNA was almost resistant to these enzymes, though a significant amount (10%) was cleaved by BglI. These results suggest that the origin is the site unfavorable for nucleosome phasing although the region can be almost completely covered with nucleosomes at higher histone : DNA ratios. The fraction of chromatin immunoprecipitated with anti-T serum after in vitro T antigen binding also decreased with increase in the histone : DNA ratios. The results suggest that T antigen binds preferentially to the internucleosomal region. T antigen preferentially bound to the chromatin reconstituted with the DNA fragment containing the origin. Inefficient phasing of nucleosomes at the origin of DNA replication may facilitate the binding of T antigen to the origin.