Galactinol synthase (GolS, EC 18.104.22.168) catalyzes formation of galactinol and the subsequent synthesis of raffinose family oligosaccharides. The relationship of GolS to drought and salt tolerance has been well documented, however, little information is available about the role of GolS gene in cold tolerance. A coding sequence of MfGolS1 cDNA was cloned from Medicago sativa spp falcata (i.e. M. falcata), a species that exhibits greater cold tolerance than alfalfa (M. sativa). MfGolS1 transcript was not detected in untreated vegetative tissues using RNA blot hybridization; however, it was greatly induced in leaves, but not in stem and petiole, after cold treatment. Higher levels of MfGolS1 transcript were induced and maintained in M. falcata than in M. sativa during cold acclimation. Accordingly, more sugars including sucrose, galactinol, raffinose and stachyose were accumulated in M. falcata than in M. sativa. The data indicated that MfGolS1 transcript and its resultant sugar accumulation were associated with the differential cold tolerance between M. falcata and M. sativa. MfGolS1 transcript was weakly induced by dehydration and salt stresses, but not responsive to abscisic acid. MfGolS1 could be induced by myo-inositol, which is proposed to participate in cold-induced MfGolS1 expression. Overexpression of MfGolS1 in tobacco resulted in elevated tolerance to freezing and chilling in transgenic plants as a result of enhanced levels of galactinol, raffinose and stachyose. Tolerance to drought and salt stresses was also increased in the transgenic tobacco plants. It is suggested that MfGolS1 plays an important role in plant tolerance to abiotic stresses.