Previous work with model transgenic plants has demonstrated that cellular accumulation of mannitol can alleviate abiotic stress. Here, we show that ectopic expression of the mtlD gene for the biosynthesis of mannitol in wheat improves tolerance to water stress and salinity. Wheat (Triticum aestivum L. cv Bobwhite) was transformed with the mtlD gene of Escherichia coli. Tolerance to water stress and salinity was evaluated using calli and T2 plants transformed with (+mtlD) or without (mtlD) mtlD. Calli were exposed to 1.0 MPa of polyethylene glycol 8,000 or 100 mM NaCl. T2 plants were stressed by withholding water or by adding 150 mM NaCl to the nutrient medium. Fresh weight of mtlD calli was reduced by 40% in the presence of polyethylene glycol and 37% under NaCl stress. Growth of +mtlD calli was not affected by stress. In mtlD plants, fresh weight, dry weight, plant height, and flag leaf length were reduced by 70%, 56%, 40%, and 45% compared with 40%, 8%, 18%, and 29%, respectively, in +mtlD plants. Salt stress reduced shoot fresh weight, dry weight, plant height, and flag leaf length by 77%, 73%, 25%, and 36% in mtlD plants, respectively, compared with 50%, 30%, 12%, and 20% in +mtlD plants. However, the amount of mannitol accumulated in the callus and mature fifth leaf (1.7-3.7 µmol g1 fresh weight in the callus and 0.6-2.0 µmol g1 fresh weight in the leaf) was too small to protect against stress through osmotic adjustment. We conclude that the improved growth performance of mannitol-accumulating calli and mature leaves was due to other stress-protective functions of mannitol, although this study cannot rule out possible osmotic effects in growing regions of the plant.