We studied the possible involvement of the GABA shunt in the plant response to low temperatures by monitoring GABA levels in barley and wheat seedlings during cold acclimation and freezing response. In frost-resistant barley seedlings, cold acclimation caused a significant increase in amino acid pools, and induced the expression of the GABA shunt genes. As a consequence, GABA was found accumulated to a higher extent during the subsequent exposure to freezing temperatures. A different picture was obtained with a frost-sensitive genotype, in which GABA accumulation occurred during the stress as well, but the activation of the GABA shunt seemed not to take place, and the substrate glutamate was almost depleted. Analogous results were found in frost-resistant and frost-sensitive wheat cultivars. Feeding non-hardened plants with exogenous glutamate resulted in increased GABA accumulation under low temperature. The overall evidences suggest that GAD activity might contribute to frost tolerance in acclimated plants.