Technical Abstract: Understanding ecosystem carbon dioxide flux requires knowledge of component fluxes including photosynthetic uptake and respiratory loss. Experimental separation of soil respiration into its heterotrophic and autotrophic components 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 13**C between C**3 and C**4 plant species to separate microbial respiration from C**4 derived soil organic matter from root respiration by C**3 forage species. Respiratory flux and carbon isotope data were collected in May, July, and September, 2008 and 2009 from plots containing either orchardgrass or white clover. The site had been a big bluestem field for about 30 years prior to establishing the orchardgrass and white clover. Thus, the soil organic C had a strong C**4 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. Root respiration accounted for about 50% of total soil respiration in July and September but only 40% in May. Root respiration under orchardgrass averaged 50% of total soil respiration compared with 43% under white clover. Additional data collected during the summer of 2010 will quantify root respiration throughout the regrowth cycle.