Technical Abstract: Understanding the regulation of ecosystem carbon dioxide flux from forage production systems requires knowledge of component fluxes, including photosynthetic uptake and respiratory loss. Experimental separation of soil respiration into its heterotrophic (free-living soil organisms) and rhizosphere components (roots and associated organisms) 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 carbon 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 gerardii, Vitman) field for about 30 years prior to establishing the orchardgrass and white clover. Thus, the soil organic C had a strong C4 signature. At each sampling period, respiration measurements were made at the mid-point of the regrowth cycle, two weeks following defoliation to a stubble height of 7 cm. Rhizosphere respiration accounted for about 50% of total 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.