A strain of the yeast Saccharomyces cerevisiae coding for KHR on the chromosome secreted a toxin that kills sensitive yeasts. The transformants of multicopy vectors carrying the KHR gene could secrete 3-4-fold the killer toxin of the donor strain. This toxic substance was purified 80-fold in specific activity from the culture filtrate by gel filtration and hydrophobic column chromatography. The purified toxin gave a single protein band with molecular mass of 20 kDa on SDS-PAGE and had an isoelectric point of pH 5.3. The toxin had novel killer activity against Candida glabrata and S. cerevisiae, but did not affect bacteria, fungi, or other yeasts.