A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.