This research was focused on investigation of the condition for protoplast formation and regeneration of protoplast fusion between Saccharomyces cerevisiae which has fermentation ability and Candida cariosilignicola which can grow at high temperature and utilize methanol. The results obtained were as follows; The highest production was collected in exponential growth phase. Ninety-nine% protoplast formation of C. cariosilignicola was obtained in glycin-NaOH buffer (pH10.0) containing Zymolyase 0.5mg/ml at $35^{circ}C$ for 1hr incubation. The highest regeneration was produced when protoplast wuwpension containing 0.5% soft agar in buffered 50mM $CaCl_{2}$ was poured as a soft overlay onto 2% agar plates. Equal amuont of protoplast suspension of two strains was mixed and centrifuged. The subsequent pellet was added to 2ml of 35% polyethylene glycol (MW 4,000) containing 50mM $CaCl_{2}$, and incubated at $30^{circ}C$ for 10min. Then 0.1ml of the suspension of aggregated protoplast was immediately covered with minimal medium and incubated at $40^{circ}C$ for 5-7 days. As results, $SC_{1}$, $SC_{2}$, and $SC_{3}$ fusants were obtained. The physiological characteristics of fusants produced by protoplast fusion were; $SC_{1}$, and $SC_{2}$ utilized maltose, galactose, methanol, potassium nitrate. $SC_{3}$ utilized all the above materials except galactose.