Restrictions to photosynthesis can limit plant growth at high temperature in a variety of ways. In addition to increasing photorespiration, moderately high temperatures (35–42 °C) can cause direct injury to the photosynthetic apparatus. Both carbon metabolism and thylakoid reactions have been suggested as the primary site of injury at these temperatures. In the present study this issue was addressed by first characterizing leaf temperature dynamics in Pima cotton (Gossypium barbadense) grown under irrigation in the US desert south-west. It was found that cotton leaves repeatedly reached temperatures above 40 °C and could fluctuate as much as 8 or 10 °C in a matter of seconds. Laboratory studies revealed a maximum photosynthetic rate at 30–33 °C that declined by 22% at 45 °C. The majority of the inhibition persisted upon return to 30 °C. The mechanism of this limitation was assessed by measuring the response of photosynthesis to CO2 in the laboratory. The first time a cotton leaf (grown at 30 °C) was exposed to 45 °C, photosynthetic electron transport was stimulated (at high CO2) because of an increased flux through the photorespiratory pathway. However, upon cooling back to 30 °C, photosynthetic electron transport was inhibited and fell substantially below the level measured before the heat treatment. In the field, the response of assimilation (A) to various internal levels of CO2 (Ci) revealed that photosynthesis was limited by ribulose-1,5-bisphosphate (RuBP) regeneration at normal levels of CO2 (presumably because of limitations in thylakoid reactions needed to support RuBP regeneration). There was no evidence of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) limitation at air levels of CO2 and at no point on any of 30 A–Ci curves measured on leaves at temperatures from 28 to 39 °C was RuBP regeneration capacity measured to be in substantial excess of the capacity of Rubisco to use RuBP. It is therefore concluded that photosynthesis in field-grown Pima cotton leaves is functionally limited by photosynthetic electron transport and RuBP regeneration capacity, not Rubisco activity.