Glandless upland cotton (Gossypium hirsutum L. cv. Coker 100A) plants were subjected to heat stress in a growth cabinet. Carbon exchange rate (CER), ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (rubisco) activation state, and RuBP and 3-phosphoglycerate (3-PGA) content were monitored. Air temperature increases from control (28°C) values were in 2.5°- to 5°-steps to 45°C, with a 1-h equilibration at each step with high ambient humidity (~75%) to allow leaf temperature to approach that of the air. Samples taken from young, fully-expanded leaves indicated that rubisco activation state (mediated by the presence of active, non-heat-denatured rubisco activase) began decreasing when leaf temperature was raised above 35°C and decreased to 25% of control values at 45°C. This was mirrored by decreases in net photosynthetic rate (CER) to 61% of control values at 42.5°C, and by a 50% decrease in the rubisco product 3-PGA, starting at 40°C and reaching a maximum at 45°C. Additionally, these changes in rubisco activity, activation state and 3-PGA content were reversible by subsequently lowering the temperature of the heat-stressed leaves. Fluorescence measurements indicated that increasing leaf temperature adversely affected flux through the photosynthetic electron transport chain. Cumulatively, these results complement a previous study (Feller et al. (1998) Plant Physiol 116, in press) demonstrating the importance of rubisco activase in the maintenance of maximal rubisco activity at relatively high temperatures.