The role of S in legume growth, N uptake, and N2 fixation was investigated using white clover (Trifolium repens L.) as a model species. We examined whether the effect of sulphate addition on N fixation resulted from a stimulation of host plant growth, a specific effect of S on nodulation, or a specific effect of S on nodule metabolism. Clones of white clover, inoculated with Rhizobium leguminosarum, were grown for 140 d in a hydroponic system with three levels of sulphate concentration (0 mM, 0.095 mM, and 0.380 mM). Nodule morphological and biochemical traits, such as root length, nodule biomass and volume, nodule protein contents (nitrogenase and leghaemoglobin obtained by an immunological approach), and root amino acid concentrations, were used to analyse the effect of sulphate availability on N2 fixation. The application of sulphate increased whole plant dry mass, root length, and nodule biomass, expressed on a root-length basis. N uptake proved less sensitive than N2 fixation to the effects of S-deficiency, and decreased as a consequence of the lower root length observed in S-deficient plants. N2 fixation was drastically reduced in S-deficient plants as a consequence of a low nodule development, but also due to low nitrogenase and leghaemoglobin production. This effect is likely to be due to down-regulation by a N-feedback mechanism, as, under severe S-deficiency, the high concentration of whole plant N and the accumulation of N-rich amino acids (such as asparagine) indicated that the assimilation of N exceeded the amount required for plant growth.