Excess of ammonia generates oxidative and osmotic stress, and results in an accumulation of compatible solutes. The aim of this study was to investigate the physiological significance of excess ammonium-induced proline and sucrose accumulation on antioxidative activity and osmotic adjustment. The detached leaves of white clover (Trifolium repense L.) were fed with 0, 10, 50, 100, and 200 mM NH4Cl, and the contribution of proline and sucrose to osmotic adjustment and their relationship with antioxidative enzymes activity were assessed. A gradual decline of relative water content and osmotic potential (Ψπ) with increasing NH4Cl feeding level was accompanied by an increase in ammonia concentration. Significant accumulation of proline and sucrose was observed when NH4Cl was fed over 100 mM compared with control (0 mM NH4Cl). The increase in enzyme activity was significant only at 200 mM for ascorbate peroxidase (APOD) and over 100 mM NH4Cl for guaiacol peroxidase (GPOD) and catalase (CAT). The contribution of proline and sucrose to osmotic adjustment over 100 mM, where proline and sucrose accumulation was more important, maintained at control levels or significantly decreased. The content of proline and sucrose as affected by NH4Cl feeding level was positively related with the activity of APOD, GPOD, and CAT. These results suggest that proline and sucrose accumulation induced by the excess of ammonium has a more influential role in antioxidative activity rather than osmotic adjustment. © 2013 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.