The Zygosaccharomyces rouxii plasmid pSB3 is one of the yeast plasmids resembling 2-microns DNA. The presence of a cis-acting locus needed for stable maintenance of pSB3 has been expected by analogy with the functions encoded by other yeast plasmids such as 2-microns DNA and pSR1. We found such a locus and designated it STB-SB3. The STB-SB3 locus was delimited within 371 bp in a unique region of pSB3. This region is included in the plasmid region where no transcript was detected. No prominent feature of the nucleotide sequence was found in this region except for a direct repeat with 65% homology consisting of 27 bp and 29 bp. This is in clear contrast with the structure of the STB locus of 2-microns DNA which has five and a half tandem repeats consisting of a highly homologous 62 bp sequence. By analyzing the sites hypersensitive to nucleases using partially purified nuclei containing pSB3, the STB-SB3 locus was found not to be tightly folded into a nucleosome structure.