For the transformation of the yeast Saccharomyces kluyveri, ura3 mutants were obtained by 5-fluoro-orotic acid selection. By utilizing the method based on treatment of intact cells with alkali cations, the ura3 strains of S. kluyveri were transformed by Saccharomyces cerevisiae-based plasmids. In the transformed cells, a S. cerevisiae centromere-based plasmid was stably replicated autonomously. Thus, this system will permit the study of gene expression and its regulation in S. kluyveri in relationship to that in S. cerevisiae.