Clotrimazole-resistant mutants from various sake yeasts show improved fermentative activity in sake mash while retaining their parental advantages for sake making. These mutants also exhibit pleiotropic drug resistance (PDR) phenotypes. To investigate the relationship between the improvement of fermentative activity and PDR phenotypes, a PDR1 mutant allele (pdr-h176) encoding a transcription factor was cloned from a clotrimazole-resistant mutant, HL176 (MATa/MAT alpha), using PCR amplification. The nucleotide sequences of pdr1-h176 and its wild allele were determined. The mutant allele contained a missense point mutation (L309S) that can confer a PDR phenotype on yeast. This amino acid substitution is located in the conserved motif II in the inhibitory domain of Pdr1p, and is very close to the cluster of three mutation points (P298A, K302Q, and M308I) described by Carvajal ct al. (Mol. Gen. Genet., 256, 406-415, 1997) in laboratory strains. A PDR1 wild allele of HL163, the parent strain of HL176, was replaced hy pdr1-h176 using gene recombination at the homologous site. The resultant transformants (PDR1/pdr1-h176) showed the same PDR phenotype as HL176, and they fermented sake mash efficiently even in the final fermentation stage, while HL163 did not. The amino acid substitution (L309S) in pdr1-h176 was considered to be sufficient to improve the fermentative activity of sake yeast, in addition to conferring the PDR phenotype.