In vivo room temperature chlorophyll a fluorescence coupled with CO2 and 02 exchange was measured to determine photosynthetic limitation(s) for spring and winter wheat (Triticum aestivum L.) grown at cold-hardening temperatures (50C/50C, day/night).Plants of comparable physiological stage, but grown at nonhardening temperatures (20°C/160C, day/night) were used in comparison. Winter wheat cultivars grown at 50C had light-saturated rates of CO2 exchange and apparent photon yields for CO2 exchange and 02 evolution that were equal to or greater than those of winter cultivars grown at 200C. In contrast, spring wheat cultivars grown at 50C showed 35% lower apparent photon yields for CO2 exchange and 25% lower light-saturated rates of CO2 exchange compared to 200C grown controls. The lower CO2 exchange capacity is not associated with a lower efficiency of photosystem 11 activity measured as either the apparent photon yield for 02 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 CO2 exchange capacity of the spring cultivars developed following long-term exposure to low temperature and did not occur following overnight exposure of nonhardened plants to 50C. Attainment of maximum freezing resistance by cold-tolerant cereals such as winter rye and winter wheat (Triticum aestivum L.) is dependent upon growth and development during prolonged exposure of seedlings to low, nonfreezing temperatures (0-5°C) prior to onset of freezing conditions. Growth and development at low temperature are therefore prerequisites for the expression of freezing resistance in these cold-tolerant cereals (8,14,17). Because photosynthesis provides the energy for this growth and development, we are interested in understanding the mechanisms by which overwintering cereals maintain optimal photosynthetic capacity at low, cold-hardening growth temperatures.The most extensive work on the effects of prolonged exposure to low, nonfreezing (0-5°C) as well as freezing temperatures on photosynthesis in cold-tolerant plants has been carried out in conifers (21,22,24 photoinhibition at low temperatures (23) similar to that observed for spinach (32), and are able to maintain light-and C02-saturated rates of photosynthesis equivalent to or higher than plants grown at 20°C (12). To exhibit these characteristics, winter rye must develop at low temperatures. Mature leaves developed at 20°C and subsequently exposed to low temperatures for up to 25 d do not exhibit these photosynthetic traits (23). Similarly, Lolium temulentum (27) and dicotyledonous winter annuals (4, 29) have equivalent rates of CO2 exchange under cold-hardening and nonhardening conditions. In contrast, when pines are exposed to coldhardening conditions they become photosynthetically inhibited (21, 24).Much of our present knowledge of cold acclimation and freezing tolerance has been gained through comparative studies...