Positioning of nucleosomes regulates the access of DNA binding factors to their consensus sequences. Nucleosome positions are determined, at least in part by the effects of DNA sequence during nucleosome assembly. Nucleosomes can also be repositioned (moved in cis) by ATP-dependent nucleosome remodeling complexes. Most studies of repositioning have used short DNA fragments containing a single nucleosome. It is difficult to use this type of template to analyze the role of DNA sequence in repositioning, however, because the many remodeling complexes are strongly influenced by nearby DNA ends. Mononucleosomal templates also cannot provide information about how repositioning occurs in the context of chromatin, where the presence of flanking nucleosomes could limit repositioning options. This protocol describes a newly developed method that allows the mapping of nucleosome positions (with and without remodeling) on any chosen region of a plasmid polynucleosomal template in vitro. The approach uses MNase digestion to release nucleosome-protected DNA fragments, followed by restriction enzyme digestion to locally unique sites, and Southern blotting, to provide a comprehensive map of nucleosome positions within a probe region. It was developed as part of studies which showed that human remodeling enzymes tended to move nucleosomes away from high affinity nucleosome positioning sequences, and also that there were differences in repositioning specificity between different remodeling complexes.