Photosynthetic potential of isolated chloroplasts was investigated during in situ water deficits. An eight day stress cycle imposed on spinach plants reduced leaf ψw by 0.57MPa, and leaf ψπ by 0.50MPa, resulting in partial turgor maintenance during the stress cycle. Pressure/volume curves confirmed the occurrence of osmotic adjustment. Leaf ψπ depression was associated with an altered response of chloroplasts to low ψπ in vitro. Optimum reaction medium ψπ for photosynthesis shifted from -1.04 to -1.57MPa, and low ψπ was not as inhibitory to photosynthesis of plastids pre-exposed to stress in situ. These data indicate that chloroplasts acclimate to low external ψπ in response to leaf water deficits. This response was still evident four days after a stress cycle ended, but was nearly reversed eight days after stress. Repeated stress cycles in situ did not increase the degree of chloroplast acclimation to low ψπ in vitro. Fast dehydration of leaves did not induce this apparent chloroplast acclimation.