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Positioning of nucleosomes has been proposed as one mechanism whereby the activity of DNA is regulated: cis-acting elements located in linker DNA might be more accessible for interaction with trans-acting protein factors than they would be if they were directly associated with histones in nucleosome core particles. The eleven base pairs constituting the autonomously replicating sequence (ARS) of the high-copy-number TRP1ARS1 plasmid of Saccharomyces cerevisiae are located in a linker region near the edge of a positioned nucleosome and form an origin of replication. Could nucleosome positioning render the ARS accessible for interaction with the proteins necessary for its function? I have tested this hypothesis by making deletions and an insertion to move the ARS into the nucleosome DNA and then examining the effects on ARS function. There is a marked decrease in copy number when the ARS is moved into the central DNA region of the nucleosome core particle, a region known to differ in structure and stability from the peripheral segments of nucleosome DNA.