Technical Abstract: It has recently been reported that high temperature slows in vivo pollen tube growth rates in Gossypium hirsutum pistils under field conditions. Although numerous physical and biochemical pollen-pistil interactions are necessary for in vivo pollen tube growth to occur, studies investigating the influence of heat-induced changes in pistil biochemistry on in vivo pollen tube growth rates are lacking. We hypothesized that high temperature would alter diurnal pistil biochemistry and that pollen tube growth rates would be dependent upon the soluble carbohydrate content of the pistil during pollen tube growth. G. hirsutum seeds were sown on different dates to obtain flowers exposed to contrasting ambient temperatures but at the same developmental stage. Diurnal pistil measurements included carbohydrate balance, adenosine triphosphate (ATP), water-soluble calcium, superoxide dismutase, glutathione reductase and NADPH oxidase. Regression analysis was used to quantify the relationship between pistil carbohydrate concentration and pollen tube growth rates. High temperature increased GR activity in the pistil only during periods not associated with in vivo pollen tube growth, and pistil ATP and water soluble calcium were unaffected by the warmer temperatures. Soluble carbohydrate levels in cotton pistils were as much as 67.5% lower under high temperature conditions (34.6°C maximum air temperature; August 4, 2009) than under cooler conditions (29.9°C maximum air temperature; August 14, 2009), and pollen tube growth rates were highly correlated with the soluble carbohydrate content of the pistil during pollen tube growth (r2 = 0.932). We conclude that moderate heat stress significantly alters diurnal carbohydrate balance in the pistil and that decreased pollen tube growth rate through the style is limited by soluble carbohydrate supply in the pistil.