The killer character of strain isolated from the main mash of sake brewing which produces a killer substance for sake yeast was transmitted to hybrids of the strain and a standard strain of Saccharomyces cerevisiae through a cytoplasmic determinant. The character was eliminated at 41 degrees C by incubation followed by growth at 30 degrees C. The killer strain produced the killer toxin in a growth-associated manner. A preparation of crude killer toxin extract showed first-order inactivation and a linear Arrhenius plot between 25 and 40 degrees C, with an activation of energy of 55.0 kcal/mol. Addition of 1% of synthetic polymer protected the toxin from inactivation by agitation but not by heat. Enhancement of the killer action toward sensitive yeast cells by only the nucleotide adenosine 5'-diphosphate (ADP) was observed after plating on agar medium as well as after incubation in liquid medium. The addition of CaCl2 reversed the enhancing effect of ADP on killing activity. This action of CaCl2 was inhibited by cycloheximide, suggesting that protein synthesis is required for recovery of toxin-induced cells in the presence of CaCl2. Further, CaCl2 overcame the decrease in the intracellular level of adenosine 5'-triphosphate (ATP) enhanced by ADP in killer-treated cells and also inhibited leakage of ATP from the cells with immediate response. The mode of killing action is discussed in terms of a transient state of the cells and the action of ADP and CaCl2.