Studies were undertaken to determine if there is an association between nonstomatally-mediated acclimation of photosynthesis to low water potential (PSI-W) and the maintenance of chloroplast volume during water stress. Spinach plants either kept well watered throughout their growth (non-acclimated), or subjected to water stress such that leaf PSI-W dropped to -1.5 megapascals (MPa) and then were rewatered (acclimated) were subjected to drought episodes. During these stress periods, photosynthesis was maintained to a greater extent in acclimated plants as compared to non-acclimated plants at PSI-W below -1 MPa. Estimates of internal leaf [CO2] suggested that photosynthetic acclimation to low PSI-W was not primarily due to altered stomatal response. As PSI-W dropped from initial values, a decline in steady state levels of ribulose 1,5-bisphosphate (RuBP) occurred in both non-acclimated and acclimated plants. RuBP decline was less severe in acclimated plants. Low PSI-W effects on chloroplast volume in non-acclimated and acclimated plants were estimated by measuring the volume of intact chloroplasts isolated from plants in solutions which were made isotonic to declining leaf osmotic potential during the drought episodes. Chloroplast volume was maintained to a greater extent at low PSI-W in acclimated, as compared with non-acclimated plants. Although substantial osmotic adjustment occurred in both non-acclimated and acclimated plants, the extent of osmotic adjustment was the same. These data were interpreted as supporting the hypothesis that cellular-level acclimation to low PSI-W is associated with chloroplast volume maintenance, and this physiological acclimation is correlated with enhanced photosynthetic capacity of the leaf at low PSI-W.