Carbohydrate metabolism was investigated in barley leaves subjected to drought or osmotic stress induced by sorbitol incubation. Both drought and osmotic stress resulted in accumulation of hexoses, depletion of sucrose and starch, and 5–10-fold increase in the level of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P₂). These changes were paralleled by an increased activity ratio of fructose-6-phosphate,2-kinase / fructose-2,6-bisphosphatase (F2KP). The drought-induced changes in carbohydrate content and Fru-2,6-P₂ metabolism were reversed upon re-watering. This reveals a reversible mechanism for modification of the F2KP enzyme activity. This suggests that F2KP might be involved in altering carbohydrate metabolism during osmotic stress. However, labelling with [¹⁴C]-CO₂ showed that sucrose synthesis was not inhibited, despite the increased Fru-2,6-P₂ levels, and demonstrated that increased flux into the hexose pools probably derived from sucrose hydrolysis. Similar effects of osmotic stress were observed in leaf sections incubated in the dark, showing that the changes did not result from altered rates of photosynthesis. Metabolism of [¹⁴C]-sucrose in the dark also revealed increased flux into hexoses and reduced flux into starch in response to osmotic stress. The activities of a range of enzymes catalysing reactions of carbohydrate metabolism in general showed only a marginal decrease during osmotic stress. Therefore, the observed changes in metabolic flux do not rely on a change in the activity of the analysed enzymes. Fructose-2,6-bisphosphate metabolism responds strongly to drought stress and this response involves modification of the F2KP activity. However, the data also suggests that the sugar accumulation observed during osmotic stress is mainly regulated by another, as yet unidentified mechanism.