A novel glucosinolate, 3-(hydroxymethyl)pentylglucosinolate, was present at high levels in Cardamine pratensis L. from eastern North America and in commercially obtained seeds, but not in C. pratensis plants from southern Scandinavia. Glucosinolates in a number of accessions of C. pratensis included glucosinolates with the side chains 1-methylethyl, 1-(hydroxymethyl)ethyl, 1-methylpropyl, 1-(hydroxymethyl)propyl, 3-methylpentyl, 3-(hydroxymethyl)pentyl, benzyl, 4-hydroxybenzyl, 4-methoxybenzyl, indol-3-ylmethyl (as well as its 1-methoxy, 4-hydroxy, and 4-methoxy derivatives) and the rare side chain 1,4-dimethoxyindol-3-ylmethyl. Substantial variation was observed for four biosynthetic characters: (i) extent of chain elongation of Ile-derived glucosinolates; (ii) biosynthesis of Phe/Tyr-derived glucosinolates in general; (iii) hydroxylation of branched-chain glucosinolates; and (iv) O-methylation of 4-hydroxybenzylglucosinolate (sinalbin). Cytological analysis of pollen mother cells and root tip cells in meiosis and mitosis established the chromosome number to be 2n = 30 for all accessions, irrespective of glucosinolate profile.