We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.