AbstractIncreasing concentrations of atmospheric carbon dioxide (CO2) may influence plant‐water relations in natural and agricultural ecosystems. A tallgrass prairie near Manhattan, KS, was exposed to elevated atmospheric CO2 using open‐top chambers (OTCs). Heat balance sap flow gauges were used to measure transpiration in ironweed [Vernonia baldwini var. interior (Small) Schub.], a C3 forb, and on individual grass culms of big bluestem (Andropogon gerardii Vitman) and indiangrass [Sorghastrum nutans (L.) Nash], both C4 grasses, in each of three treatments: (i) CE (chamber enriched, 2× ambient CO2); (ii) CA (chamber ambient, no CO2 enrichment); and (iii) NC (no chamber, no CO2 enrichment). Sap flow data were coupled with measurements of stomatal conductance, plant/canopy resistance, and whole‐chamber evapotranspiration (ET) to determine the effect of elevated CO2 on water use at different scales. Because of frequent rainfall during the study, all data were collected under well‐watered conditions. Comparisons of CE and CA showed that sap flow was reduced by 33% in ironweed, 18% in big bluestem, and 22% in indiangrass under CO2 enrichment. Whole‐chamber ET was reduced by 23 to 27% under CO2 enrichment. Comparisons of CA and NC showed that the environmental effect of the OTCs caused a 21 to 24% reduction in transpiration. Stomatal conductance decreased from 7.9 to 3.6 mm s−1 in big bluestem and from 5.3 to 3.2 mm s−1 in indiangrass under CO2 enrichment. Soil water was consistently highest under elevated CO2, reflecting the large reductions in transpiration. During sap flow measurements, whole‐plant stomatal resistance to water vapor flux in big bluestem increased from 103 to 194 s m−1 under elevated CO2.