Stable isotopes of carbon dioxide (CO 2 ) can be used as natural tracers to better understand carbon cycle processes and exchange pathways between the biosphere and atmosphere. In this study, we used a tunable diode laser (TDL) technique for continuous fast measurement of the stable isotopomers C 18 O 16 O and C 16 O 2 and their ratio, δ a . The TDL system was configured to measure mixing ratios of [C 16 O 2 ] and [C 18 O 16 O] at wavenumber frequencies of 2308.225 and 2308.416cm −1 , respectively. Two-minute precision (1standard deviation) was 0.0004, 0.09μmolmol −1 , and 0.26‰ for [C 18 O 16 O], [C 16 O 2 ] and δ a , respectively. Comparison of TDL and mass spectrometry flask measurements showed relatively good agreement (r 2 =0.94) with a standard deviation of 0.55‰ for the residual values. Estimates of the isotope signature of ecosystem flux components over a soybean (Glycine max) field were examined. These data represent one of the first continuous flux measurements of C 18 O 16 O. The isotope signature of net ecosystem CO 2 exchange at night ranged from −15 to −7‰ and during the daytime from −40 to −20‰. A simple estimate of canopy-scale photosynthetic discrimination showed significant diurnal variation and averaged 10.5‰ (±8.8‰). The large difference between the isotope signature of respiration and midday canopy photosynthesis represented significant isotopic disequilibrium. Coupled with recent advances in measuring water vapor isotopomers with the TDL technique, a new opportunity is emerging to better understand the dynamics, complex interactions, and discrimination mechanisms controlling land-atmosphere C 18 O 16 O exchange.