The effect of osmotic dehydration on metabolic reactions in three different subcellular compartments (chloroplast, cytoplasm and mitochondria) was studied in vacuum-infiltrated thin leaf slices from various plants, in the absence of stomatal control. The reactions tested were CO2 fixation in the light (chloroplast), CO2 fixation in the dark (cytoplasm), and O2 uptake in the dark (mitochondria). In most plants, the sensitivity of dark CO2 fixation to dehydration was similar to the sensitivity of photosynthesis. In leaf slices from a plant with Crassulacean acid metabolism (Kalanchoe pinnata), dark CO2 fixation (which reached similar rates as light fixation) was slightly more sensitive to osmotic stress than photosynthesis. Dark respiration (measured as O2 uptake) was significantly more resistant to hypertonic stress than both types of CO2 fixation. In crude leaf extracts from spinach, the response of soluble enzymes from the three different subcellular compartments to high concentrations of various electrolytes and neutral compounds was examined and compared with the in-vivo data.