The hypoglycemic drug tolbutamide is commonly used as a probe drug to evaluate CYP2C9 enzyme activity in terms of production of 4'-hydroxytolbutamide. In the present study, an initial screening of seven filamentous fungi was carried out to identify which was most competent to transform tolbutamide into 4'-hydroxytolbutamide. From this screening, the fungus Cunninghamella blakesleeana AS 3.910 was selected as a suitable bioconverter. At a concentration of 1.2 mg ml(-1), the growing fungus transformed 95.0% of tolbutamide into 4'-hydroxytolbutamide in 96 h. With resting culture, the yield could reach 91.7% and exceeded 91.0% even when the tolbutamide concentration was increased to 4.0 mg ml(-1). On scale-up to 3 l buffer containing 12.0 g tolbutamide, 90% of tolbutamide was transformed into 4'-hydroxytolbutamide in 96 h. Work-up of the broth by column chromatography and recrystallization yielded 6.5 g (53.9% recovered) of 4'-hydroxytolbutamide with a purity of more than 99%. These results suggest C. blakesleeana AS 3.910 is a useful biosynthetic tool in the preparation of 4'-hydroxytolbutamide.