The nucleotide sequence of a 6251 base-pair plasmid, pSR1, harbored in an osmophilic haploid yeast, Zygosaccharomyces rouxii (formerly Saccharomyces rouxii), was determined. No homology was detected between the sequences of pSR1 and 2-micron DNA of Saccharomyces cerevisiae. pSR1 has a pair of inverted repeats consisting of completely homologous 959 base-pair sequences, which separate two unique sequences 2654 base-pairs and 1679 base-pairs long. Each inverted repeat has an ARS sequence functional in both Z. rouxii and S. cerevisiae hosts. Short direct repeats or dyad symmetries were observed in the inverted repeats similar to those found close to the replication origin of 2-micron DNA. Three open reading frames, P, S and R, each able to encode a protein of molecular weight larger than 10,000, were found. Insertional inactivation of R gave rise to a defect in the intramolecular recombination at the inverted repeats, and that of S reduced the copy number of pSR1 in the S. cerevisiae host. The maintenance stability of the plasmid was also tested in the heterogeneous S. cerevisiae host, but the results of the insertional inactivation of P, S and R were ambiguous. pSR1 and 2-micron DNA were compatible in S. cerevisiae cells, but the protein factors encoded by these plasmids did not complement each other.