The mixed effects of nitrogen nutrition and sulphate assimilation were investigated in barley plants (Hordeum vulgare var. Alfeo) that were subjected to long-term sulphur and/or nitrogen starvation, by measuring the O-acetylserine(thio)lyase (OASTL–EC 4.2.99.8) activity, changes in –SH compounds and amino acid levels.The growth of barley plants cultured in the hydroponic vessels was severely affected by altered nutrient levels. The barley plants grown in medium deprived of nitrogen and/or sulphur sources for 21 days showed increase in both root length and weight. In contrast, the shoot growth was reduced in nitrogen-starved plants and was unaffected by sulphur deprivation. Sulphur starvation affected the level of proteins in barley plants more than nitrogen deprivation. The decline in the protein levels observed under sulphur-deficient conditions was coupled with the accumulation of glutamine, asparagine and serine, mainly in the roots; additionally, a nitrogen deficiency in the roots promoted a decrease in both glutathione and cysteine levels.The simultaneous deprivation of nitrogen and sulphur in plants leads to an alteration in their metabolism; high levels of glutathione (GSH) in the shoots could signify the induction of a mechanism intended for coping with stressful conditions.Sulphate deprivation enhanced OASTL activity, mainly in the roots; on the other hand, OASTL increases observed under S deprivation were clearly dependent on the nitrogen availability in the culture medium. In fact, the nitrate supply to the N and S starved plants that showed OASTL activity very low, rapidly recovered the OASTL activities to the levels typical of control plants. Nevertheless, the ammonium supply had negligible effects on the OASTL activity only observed after three days in the roots.Our results support the hypothesis that in barley plants, a portion of S assimilation (up to cysteine biosynthesis) occurs in the roots, and a reciprocal influence of nitrogen assimilation on cysteine synthesis occurs.