The conditions of endogenous hydrolysis of glucotropaeolin to bioactive benzyl isothiocyanate for T. majus hairy root cultures giving high yields of glucotropaeolin and myrosinase were optimized. After in vitro glucotropaeolin hydrolysis at pH 5.5, optimal for myrosinase activity, 54% of glucotropaeolin were converted to benzyl isothiocyanate, 22% to benzyl cyanide, and 1.9% to benzyl thiocyanate. During endogenous glucotropaeolin hydrolysis, maximal benzyl isothiocyanate yield, 87% of tissue glucotropaeolin, was detected at pH 7.5, in the 60th min of incubation. In the presence of 100 M ascorbic acid, the rate of hydrolysis increased and in the 60th min 99% of glucotropaeolin was converted to benzyl isothiocyanate. The highest benzyl isothiocyanate yield, 0.746 mmol/10 g fresh weight, was obtained during endogenous hydrolysis at pH 7.5, in the presence of 100 M ascorbic acid from the hairy roots with glucotropaeolin content, enhanced by precursors (phenylalanine + cystein) and inhibitor of phenylalanine ammonia-lyase activity (l-1-amino-2-phenylethylphosphonic acid).