AbstractClear influence of N and S applications on the profile of individual glucosinolates in the seeds of winter oilseed rape (Brassica napus L) was found in field experiments. The major effect of N was on the relative abundance of the four alkenyl glucosinolates. Increasing the N rate increased the relative proportion of 2‐hydroxybut‐3‐enyl at the expense of pent‐4‐enyl, and to a lesser extent, 2‐hydroxypent‐4‐enyl, in the double‐low (low erucic acid and low glucosinolates) variety Cobra, but at the expense of but‐3‐enyl in the single‐low (low erucic acid) variety Bienvenu. The results strongly suggest that a high N supply favours the hydroxylation step from but‐3‐enyl to 2‐hydroxybut‐3‐enyl. In contrast, the major effect of S appeared to be on the relative abundance of the alkenyl and indole groups as a whole. Sulphur deficiency decreased the concentrations of the alkenyl glucosinolates more than those of the indole glucosinolates, whereas S application to a S‐deficient crop resulted in a larger response in the alkenyl group than in the indole group. The more sensitive response of alkenyl glucosinolates to the plant S status is probably due to the requirement of methionine in their biosynthesis, as compared with the indole glucosinolates which are derived from tryptophan. Implications for changes in the glucosinolate profile in rapeseed are discussed.