Nucleosome ordering on a variety of replicating plasmids, assembled into chromatin in transfected COS-1 cells, was studied by micrococcal nuclease digestion of isolated nuclei. Generally, no more than three well defined multiples of a unit nucleosome repeat, which resembled the first three bands of the (187 +/- 5 base pair (bp)) cellular chromatin ladder, could be detected in constructs that contained a near-minimal SV40 replication origin. In contrast, constructs that additionally contained the SV40 early region exhibited significantly more regular nucleosome arrangements. In some cases, eight to nine multiples of a 203 +/- 5-bp repeat could be resolved. The presence of the SV40 early region was necessary for physiological nucleosome alignment over the SV40 late and ori regions, or onto adjacent pBR327 DNA. In an in vitro chromatin assembly system, using purified chicken erythrocyte histones plus polyglutamic acid, a portion of SV40 DNA became packaged into a highly ordered 200 +/- 5-bp nucleosome array, which encompassed the early region and extended for about 2800 bp. The data suggest that in the cell nucleus, nucleosome ordering on SV40 DNA might spread from sequences in the early region, close to where transcription terminates, probably as a consequence of histone H1-nucleosome interactions.