Fragments containing ARSes were cloned from the genomic DNA of the yeast Saccharomyces exiguus Yp74L-3, and the essential regions for ARSes were restricted for these fragments, Mapping studies of ARS-acting sequences in one of these fragments suggested that S. exiguus recognizes a sequence as an ARS that is different from that recognized by Saccharomyces cerevisiae. Two ARS essential regions of S. exiguus were sequenced, and an ARS con consensus sequence of S. exiguus was deduced to be MATTAMWAWWTK. This sequence differs significantly from that of S, cerevisiae in two positions, suggesting that these nucleotide substitutions cause the difference in the ARS-recognition modes between S. exiguus and S. cerevisiae.