The photosynthetic pathway composition (C(3):C(4) mixture) of an ecosystem is an important controller of carbon exchanges and surface energy flux partitioning, and therefore represents a fundamental ecophysiological distinction. To assess photosynthetic mixtures at a tallgrass prairie pasture in Oklahoma, we collected nighttime above-canopy air samples along concentration and isotopic gradients throughout the 1999 and 2000 growing seasons. We analyzed these samples for their CO(2) concentration and carbon isotopic composition and calculated C(3):C(4) proportions with a two-source mixing model. In 1999, the C(4) percentage increased from 38% in spring (late April) to 86% in early fall (mid-September). The C(4) percentages inferred from ecosystem respiration measurements in 2000 indicate a smaller shift, from 67% in spring (early May) to 77% in mid-summer (late July). We also sampled daytime CO(2 )concentration and carbon isotope gradients above the canopy to determine ecosystem discrimination against (13)CO(2) during net uptake. These discrimination values were always lower than corresponding nighttime ecosystem respiration isotopic signatures would suggest. After accounting for the isotopic disequilibria between respiration and photosynthesis resulting from seasonal variations in the C(3):C(4) mixture, we estimated canopy photosynthetic discrimination. The C(4) percentage calculated from this approach agrees with the percentage determined from nighttime respiration for sampling periods in both growing seasons. Isotopic imbalances between photosynthesis and respiration are likely to be common in mixed C(3):C(4 )ecosystems and must be considered when using daytime isotopic measurements to constrain ecosystem physiology. Given the global extent of such ecosystems, isotopic imbalances likely contribute to global variations in the carbon isotopic composition of atmospheric CO(2).