We have asked where the nucleosomes are located with respect to the replication origin and regulatory region of simian virus 40 DNA, what would be the possible functional consequences of the identified locations, and to what extent these locations correlate with the current views on mechanisms involved in establishing nucleosome-free regions in chromatin. To identify the precise location of nucleosomes, we have shot-gun cloned and sequenced nucleosomal DNA obtained from micrococcal nuclease digestion of wt776 chromatin prepared late in infection. Our results indicate that nucleosomes do not occupy unique positions over the replication origin or the elements involved in transcriptional control. However, it appears that the nucleosome distribution is not random, since several nucleosomes are represented by two or more independently generated clones. Two nearly identical cloned fragments map over the replication origin; five include 1.5 copies of the 72 base-pair enhancer sequences; and eight map to a region that spans a DNA bending locus and the major transcription initiation site of the late genes. The complex nucleosome distribution pattern observed in our direct analysis suggests that disparate nucleosome-free regions may be involved in controlling replication, and selective expression of the viral early or late genes.