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Understanding the regulation of ecosystem CO2 flux from forage production systems requires knowledge of component fluxes, including photosynthetic uptake and respiratory loss. Experimental separation of soil respiration into heterotrophic and rhizosphere respiration has been difficult, complicating efforts to quantify management and environmental effects on grazing land C sequestration. This study takes advantage of differences in the natural abundance of 13C between C3 and C4 plant species to separate microbial respiration of C4–derived soil organic matter from root respiration by C3 forage species. Respiratory flux and C isotope data were collected in May, July, and September 2008 and 2009 from plots containing either orchardgrass (Dactylis glomerata L.) or white clover (Trifolium repens L.). The site had been a big bluestem (Andropogon gerardi Vitman) field for about 30 yr before grass and clover establishment. Thus, the soil organic C had a strong C4 signature. At each sampling period, respiration measurements were made at the midpoint of the regrowth cycle, 2 wk following mowing. Rhizosphere respiration accounted for about 50% of soil respiration in July and September but only 40% in May. Rhizosphere respiration under orchardgrass averaged 50% of total soil respiration compared with 43% under white clover. Accurate quantification of the components of ecosystem respiration will aid in the development of realistic models to simulate ecosystem C flux.