We made weekly measurements of carbon (δ13C) and oxygen (δ18O) isotopes of atmospheric CO2 in a C3/C4 tallgrass prairie during the growing season for 3 years with contrasting soil moisture conditions. Air samples above and within canopies were collected using 100-ml flasks at night to characterize isotopic composition of ecosystem respiration. We used a two-source mixing line (Keeling plot) approach to estimate isotope ratios of ecosystem respired CO2 for both carbon (δ13CR) and oxygen (δ18OR). Measured net ecosystem CO2 exchange (NEE) showed the largest net carbon uptake in 2004, followed by 2003 and 2002. This interannual difference in NEE strongly depends on the amount and distribution of precipitation received by this tallgrass prairie. Precipitation also affects the timing of the seasonal transition from C3 dominance in spring to C4 dominance in summer. Variations of δ13CR showed that C4 plants dominated ecosystem respiration in 2003 and 2004, except in early spring when C3 plants were more active. In contrast, contributions of C3 plants were relatively higher for an extended period in the summer of 2002, when a severe drought occurred. Typically, C3 forbs extract water and nutrients from soil layers below that of the C4 grasses and remain photosynthetically active in periods when C4 grasses have water stress that limits photosynthesis. Drought-reduced C4 grass photosynthesis was lower than temperature-limited C3 forb growth during this period. We used an integrated isotope land surface model (ISOLSM) to simulate (and compare to measurements) net CO2 fluxes, δ18O values of leaf and soil water, and δ18O values of aboveground and soil respiration. The Keeling plot analysis becomes less reliable for estimating δ18OR values when the surface soil is dry. We suspect this is due to low CO2 production in the soil when water is limiting, in which case the invasion (abiotic) effect is more significant. ISOLSM reasonably captured seasonal variations of measured δ18OR in all 3 years, indicating the model's consistency of predicting δ18OR in different soil water conditions. Model simulations also showed that nighttime δ18O values of aboveground respiration were variable, often becoming very positive in water-stressed conditions primarily because of the low relative humidity and resultant elevated δ18O values of leaf water.