Three strains of filamentous fungi have been isolated from potato sprouts to obtain an enzyme degrading the glycoalkaloids. All of the strains hydrolyzed alpha-chaconine and not alpha-solanine when grown on the sprouts. From strain HP341, identified as Plectosphaerella cucumerina, the enzyme hydrolyzing a-chaconine was purified on columns of DEAE-Toyopearl and Phenyl-Toyopearl. The partially purified enzyme hydrolyzed alpha-chaconine to beta(1)-chaconine but not to beta(2)- or gamma-chaconine, suggesting that the enzyme is a rhamnosidase specific for the hydrolysis of the rhamnose (C-1-C-4) glucose linkage in a-chaconine. Conversion of alpha-chaconine to beta(1)-chaconine may be the first step of detoxification for filamentous fungi to grow on potato sprouts that accumulated antifungal alpha-chaconine.