We investigate the utility of an improved isotopic method to partition the net ecosystem exchange of CO2 (F) into net photosynthesis (FA) and nonfoliar respiration (FR). Measurements of F and the carbon isotopic content in air at a high‐elevation coniferous forest (the Niwot Ridge AmeriFlux site) were used to partition F into FA and FR. Isotopically partitioned fluxes were then compared with an independent flux partitioning method that estimated gross photosynthesis (GEE) and total ecosystem respiration (TER) based on statistical regressions of night‐time F and air temperature. We compared the estimates of FA and FR with expected canopy physiological relationships with light (photosynthetically active radiation) and air temperature. Estimates of FA and GEE were dependent on light as expected, and TER, but not FR, exhibited the expected dependence on temperature. Estimates of the isotopic disequilibrium D, or the difference between the isotopic signatures of net photosynthesis (δA, mean value −24.6‰) and ecosystem respiration (δR, mean value −25.1‰) were generally positive (δA>δR). The sign of D observed here is inconsistent with many other studies. The key parameters of the improved isotopic flux partitioning method presented here are ecosystem scale mesophyll conductance (gm) and maximal vegetative stomatal conductance (gcmax). The sensitivity analyses of FA, FR, and D to gcmax indicated a critical value of gcmax (0.15 mol m−2 s−1) above which estimates of FA and FR became larger in magnitude relative to GEE and TER. The value of D decreased with increasing values of gm and gcmax, but was still positive across all values of gm and gcmax. We conclude that the characterization of canopy‐scale mesophyll and stomatal conductances are important for further progress with the isotope partitioning method, and to confirm our anomalous isotopic disequilibrium findings.