A high level production system for heterologous protein by cold culture of yeast transformants at 15 degrees C was developed. The yeast transformants, carrying a plasmid containing cDNA for Aspergillus oryzae alpha-amylase (Taka-amylase A) or human lysozyme synthetic DNA, were cultivated in a selective medium for 1 or 2 days until full growth at 30 degrees C. The yeast cells were harvested by centrifugation from the culture fluid and then were transferred to YPD medium. These inoculated broths were incubated for 2 days at 15 degrees C and then for another 2 days at 30 degrees C. By the cold culture method described above, higher amounts of Taka-amylase A (28.6 mg/liter) and human lysozyme (6.1 mg/liter) were produced by the yeast transformants compared to those by conventional methods. Heterologous protein productions using YEp, YCp, and YIp types of yeast expression vectors with ADH1 or GAPDH promoter by the cold culture method showed effective productivity of about 2-fold compared to those by the conventional method of culture at 30 degrees C. The high level production of heterologous protein by this method was not specific to the S. cerevisiae strains examined.