The acclimation of C-4 photosynthesis to low temperature was studied in the montane grass Muhlenbergia montana in order to evaluate inherent limitations in the C-4 photosynthetic pathway following chilling. Plants were grown in growth cabinets at 26 degreesC days, but at night temperatures of either 16 degreesC (the control treatment), 4 degreesC for at least 28 nights (the cold-acclimated treatment), or 1 night (the cold-stress treatment). Below a measurement temperature of 25 degreesC, little difference in the thermal response of the net CO2 assimilation rate (A) was observed between the control and cold-acclimated treatment. By contrast, above 30 degreesC, A in the cold-acclimated treatment was 10% greater than in the control treatment. The temperature responses of Rubisco activity and net COP assimilation rate were similar below 22 degreesC, indicating high metabolic control of Rubisco over the rate of photosynthesis at cool temperatures. Analysis of the response of A to intercellular CO2 level further supported a major limiting role for Rubisco below 20 degreesC. As temperature declined, the CO2 saturated plateau of A exhibited large reductions, while the initial slope of the CO2 response was little affected. This type of response is consistent with a Rubisco limitation, rather than limitations in PEP carboxylase capacity. Stomatal limitations at low temperature were not apparent because photosynthesis was CO2 saturated below 23 degreesC at air levels of CO2. In contrast to the response of photosynthesis to temperature and COP in plants acclimated for 4 weeks to low night temperature, plants exposed to 4 degreesC for one night showed substantial reduction in photosynthetic capacity at temperatures above 20 degreesC, Because these reductions were at both high and low CO2, enzymes associated with the C-4 carbon cycle were implicated as the major mechanisms for the chilling inhibition. These results demonstrate that C-4 plants from climates with low temperature during the growing season can fully acclimate to cold stress given sufficient time. This acclimation appears to involve reversal of injury to the C-4 cycle following initial exposure to low temperature. By contrast, carbon gain at low temperatures generally appears to be constrained by the carboxylation capacity of Rubisco, regardless of acclimation time. The inability to overcome the Rubisco limitation at low temperature may be an inherent limitation restricting C-4 photosynthetic performance in cooler climates.