Stable isotopes of CO2 are used as natural tracers to better understand carbon cycle processes and exchange pathways between the biosphere and atmosphere. In this paper we examine isotopic CO2 fluxes (13CO2, C16O2 and C18O16O) measured using tunable diode laser (TDL) spectroscopy and micrometeorological methods over a corn-soybean rotation ecosystem in the Upper Midwest, United States to: 1) partition net ecosystem CO2 exchange into photosynthesis and respiration; 2) estimate the relative contributions of heterotrophic and autotrophic respiration; and 3) determine the seasonal and interannual variation in the isotopic composition of respiration and photosynthetic discrimination. Methodological limitations and future directions are discussed.