Technical Abstract: The North American tallgrass prairie is composed of a diverse mix of C3 and C4 plant species that are subject to multiple resource limitations. C4 grasses dominate this ecosystem, purportedly due to greater photosynthetic capacity and resource use-efficiency associated with C4 photosynthesis. We tested the hypothesis that intrinsic physiological differences between C3 and C4 species are consistent with C4 grass dominance by comparing leaf gas exchange and chlorophyll fluorescence variables for 7 C4 and C3 herbaceous species in two different settings: experimental microcosms with abundant resources and natural grassland sites with more limited resources. In the microcosms, C4 grasses had higher photosynthetic rates (Amax at ambient Ca), water potentials and water use efficiency than the C3 species. These differences were absent in the field, where several variables suggested that N limitation reduced photosynthetic rates in all non-leguminous species. Thus, intrinsic photosynthetic advantages for C4 species measured in resource-rich microcosms could not explain the dominance of C4 species in the field. Instead, C4 dominance in this ecosystem may depend more on the ability of the grasses to grow rapidly when resources are plentiful and to tolerate multiple limitations when resources are scarce.