In vivo room temperature chlorophyll a fluorescence coupled with CO(2) and O(2) exchange was measured to determine photosynthetic limitation(s) for spring and winter wheat (Triticum aestivum L.) grown at cold-hardening temperatures (5 degrees C/5 degrees C, day/night). Plants of comparable physiological stage, but grown at nonhardening temperatures (20 degrees C/16 degrees C, day/night) were used in comparison. Winter wheat cultivars grown at 5 degrees C had light-saturated rates of CO(2) exchange and apparent photon yields for CO(2) exchange and O(2) evolution that were equal to or greater than those of winter cultivars grown at 20 degrees C. In contrast, spring wheat cultivars grown at 5 degrees C showed 35% lower apparent photon yields for CO(2) exchange and 25% lower light-saturated rates of CO(2) exchange compared to 20 degrees C grown controls. The lower CO(2) exchange capacity is not associated with a lower efficiency of photosystem II activity measured as either the apparent photon yield for O(2) evolution, the ratio of variable to maximal fluorescence, or the level of reduced primary quinone electron acceptor maintained at steady-state photosynthesis, and is most likely associated with carbon metabolism. The lower CO(2) exchange capacity of the spring cultivars developed following long-term exposure to low temperature and did not occur following over-night exposure of nonhardened plants to 5 degrees C.