Photosynthesis is reduced at low leaf water potentials (Psi(l)) but repeated water deficits can decrease this reduction, resulting in photosynthetic acclimation. The contribution of the stomata and the chloroplasts to this acclimation is unknown. We evaluated stomatal and chloroplast contributions when soil-grown sunflower (Helianthus annuus L.) plants were subjected to water deficit pretreatments for 2 weeks. The relationship between photosynthesis and Psi(l), determined from gas-exchange and isopiestic thermocouple psychometry, was shifted 3 to 4 bars towards lower Psi(l), in pretreated plants. Leaf diffusive resistance was similarly affected. Chloroplast activity, demonstrated in situ with measurements of quantum yield and the capacity to fix CO(2) at all partial pressures of CO(2), and in vitro by photosystem II activity of isolated organelles, was inhibited at low Psi(l) but less in pretreated plants than in control plants. The magnitude of this inhibition indicated that decreases in chloroplast activity contributed more than closure of stomata both to losses in photosynthesis and to the acclimation of photosynthesis to low Psi(l).