Heat and drought stresses are often coincident and constitute major factors limiting global crop yields. A better understanding of plant responses to the combination of these stresses under production environments will facilitate efforts to improve yield and water use efficiencies in a climatically changing world. To evaluate photosynthetic performance under dry-hot conditions, four cotton (Gossypium barbadense L.) cultivars, Monseratt Sea Island (MS), Pima 32 (P32), Pima S-6 (S6) and Pima S-7 (S7), were studied under well-watered (WW) and water-limited (WL) conditions at a field site in central Arizona. Differences in canopy temperature and leaf relative water content under WL conditions indicated that, of the four cultivars, MS was the most drought-sensitive and S6 the most drought-tolerant. Net CO ₂ assimilation rates (A) and stomatal conductances (gs) decreased and leaf temperatures increased in WL compared to WW plants of all cultivars, but MS exhibited the greatest changes. The response of A to the intercellular CO ₂ concentration (A-C _i) showed that, along with stomatal closure, non-stomatal factors associated with heat stress also limited A under WL conditions, especially in MS. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased in WL compared to WW plants, consistent with thermal inhibition of Rubisco activase activity. The extent of Rubisco deactivation could account for the metabolic limitation to photosynthesis in MS. Taken together, these data reveal the complex relationship between water availability and heat stress for field-grown cotton plants in a semi-arid environment. Both diffusive (drought-stress-induced) and biochemical (heat-stress-induced) limitations contributed to decreased photosynthetic performance under dry-hot conditions.