In this paper we describe measurements and modeling of 18O in CO2 and H2O pools and fluxes at a tallgrass prairie site in Oklahoma. We present measurements of the δ18O value of leaf water, depth-resolved soil water, atmospheric water vapor, and Keeling plot δ18O intercepts for net soil-surface CO2 and ecosystem CO2 and H2O fluxes during three periods of the 2000 growing season. Daytime discrimination against C18OO, as calculated from measured above-canopy CO2 and δ18O gradients, is also presented. To interpret the isotope measurements, we applied an integrated land-surface and isotope model (ISOLSM) that simulates ecosystem H218O and C18OO stocks and fluxes. ISOLSM accurately predicted the measured isotopic composition of ecosystem water pools and the δ18O value of net ecosystem CO2 and H2O fluxes. Simulations indicate that incomplete equilibration between CO2 and H2O within C4 plant leaves can have a substantial impact on ecosystem discrimination. Diurnal variations in the δ18O value of above-canopy vapor had a small impact on the predicted δ18O value of ecosystem water pools, although sustained differences had a large impact. Diurnal variations in the δ18O value of above-canopy CO2 substantially affected the predicted ecosystem discrimination. Leaves dominate the ecosystem 18O-isoflux in CO2 during the growing season, while the soil contribution is relatively small and less variable. However, interpreting daytime measurements of ecosystem C18OO fluxes requires accurate predictions of both soil and leaf 18O-isofluxes.