Tomato (Lycopersicon esculentum Mill.) was used as a model system for investigating the effects of glycinebetaine applications on plants, as this species is unable to synthesise glycinebetaine, but is sensitive to exogenous supply. Glycinebetaine application significantly increased stomatal conductance of tomato plants grown in well-watered, water-deficient or saline conditions; however, glycinebetaine did not affect the leaf ABA concentration, relative water content, sap osmotic potential, or shoot water potential. Glycinebetaine applied to the foliage was readily absorbed, but the concentrations in the leaves made a negligible contribution to the total leaf sap osmotic potential. Our results indicate that applied glycinebetaine was involved in regulation of stomatal conductance but not via ABA metabolism or water relations. Glycinebetaine may have been accumulated in specific cells or cellular compartments, with consequences for stomatal functions. Further work is required to elucidate the cellular and subcellular localisation of applied glycinebetaine.