To characterize the mechanisms of amino acid accumulation under sulphur (S)-deficiency and its physiological significance in Brassica napus, stable isotopes 15N and 34S were employed. The plants were exposed for 9 days to S-deficient conditions (0.05 mM vs 1.5 mM sulphate). After 9 days of S-deficiency, leaf-osmotic potential and total chlorophyll content significantly decreased. S uptake decreased by 94%, whereas N uptake and biomass were not significantly changed. Using 15N and 34S labelling, de novo synthesis of amino acids and proteins derived from newly absorbed NO3− and SO42− and the content of N and S in the previously synthesized amino acids and proteins were quantified. At the whole plant level, S-deficiency increased the pool of amino acids but resulted in strong decrease of incorporation of newly absorbed NO3− and SO42− into amino acids by 22.2 and 76.6%, respectively, compared to the controls. Total amount of N and S incorporated into proteins also decreased by 28.8 and 62.1%, respectively. The levels of 14N- and 32S-proteins (previously synthesized proteins) strongly decreased, mainly in mature leaves. The data thus indicate that amino acid accumulation under short-term S-deficiency results from the degradation of previously synthesized proteins rather than from de novo synthesis.