As an essential previous step towards the development of cell fusion to breed a new brewing yearst strain, several factors predicted to affect the protoplast formation of S. cerevisiae, C. tropicalis and E. fibuligera were investigated in order to obtain the protoplasts in high yields. The optimum pH and temperature for the protoplas formation were 7.5 and 35$^{circ}C$, respectively. Pretreatment of the yeast cells with 2-mercaptoethanol stimulated the protoplast formation and 50mM of the reagent was found as effective. Among several osmotic stabilizers tested for their effect on protoplas formation, 0.6M KCI was comparatively favorable.