Fusion of protoplasts prepared from haploid strains of Saccharomyces yeasts having identical mating type was induced with the aid of polyethylene glycol. Stable fusion products were isolated by complementation of the auxotrophic markers. Of 64 isolates derived by protoplast fusion between two different haploid strains having alpha mating type, 35 fusion products were estimated from their cell volumes to be diploid, 13 to be triploid and 16 to be tetraploid. The isolates showing tetraploid cell size were thought to have resulted from fusion of three protoplasts of one strain and one protoplast of the other (three-to-one fusion) or from two-to-two fusion. In protoplast fusion of three different haploid strains having alpha mating type, all four possible phenotypes of fusion product were recovered. Fusion products of three different protoplasts were obtained in much lower frequency (2.1 x 10(-6)) than those of two different protoplasts (1.2 x 10(-5) to 1.4 x 10(-4)) in the three other combinations. Genetic analyses revealed that triploid fusion products were formed by protoplast fusion of two different strains as well as of three different strains.