Four indole glucosinolates were isolated from different cruciferous tissues and tissue cultures. After separation in the form of their desulfo derivatives, they were separated by HPLC and analysed by mass spectrometry. Besides 3-indolylmethylglucosinolate and 1-methoxy-3-indolylmethylglucosinolate, two new compounds were identified as 5-hydroxy-3-indolylmethylglucosinolate and 5-methoxy-3-indolylmethylglucosinolate, respectively. Feeding experiments revealed that [ring-2-14C]tryptophan was a precursor of all of these glucosinolates and that [Me-14C 1 methionine serves as the methyl donor of the methoxylated derivatives.