Cereal plants become more resistant to freezing when first exposed to a period of cold‐acclimation. Many physiological and molecular changes have been shown to occur at low temperatures, but the role and the contribution of each to frost resistance is still poorly understood. Two cultivars of barley ( Hordeum vulgare L.), the winter barley Onice and the spring barley Gitane, were acclimated under controlled conditions under an 8‐h photoperiod at 4°C (light) and 2°C (dark) for 21 days. Changes in free proline, ABA, water‐soluble carbohydrates and free fatty acids were measured to assess their involvement in cold‐acclimation and to explain the different frost‐resistant capacities of the two cultivars. Exposure of barley plants to low temperature resulted in an equal increase in proline in both cultivars. During the first days of cold acclimation, ABA levels showed a peak in the frost‐resistant cultivar, lasting about 24 h, followed by a decrease. The water soluble carbohydrates reached their highest content after 3 days of hardening, although after 14 to 21 days of acclimation the carbohydrate content was similar to that of unhardened plants. The frost‐resistant Onice had a much higher free fatty acid content than the frost‐sensitive Gitane. Furthermore in Onice 86% of free farty acids was represented by unsaturated molecular species. Inolenic acid alone being 71%. In contrast, in the frost‐sensitive cultivar only 31% of free fatty acids was unsaturated and a large amount of 9‐oxo‐nonanoic acid, a product present in the linolenic acid cascade, was also detected. The ABA content after 2 days of hardening and the free fatty acid composition were clearly different between the two cultivars and may explain, at least in part, the different frost‐resistant capacities of Onice and Gitane.