Xylophagous termites possess symbiotic bacteria that fix atmospheric nitrogen (N2). Although symbiotic N2 fixation is central to termite nutrition and ecologically important, it is energetically costly. Using stable isotopes, we tested the hypothesis that symbiotic N2 fixation would decrease in workers of the eastern subterranean termite, Reticulitermes flavipes Kollar, which were exposed to high nitrogen diets. To calculate the isotope discrimination factor occurring as a result of digestion, Δdig, and which occurs as the result of N2 fixation, Δfix, symbiotic N2 fixation was inhibited via force feeding termites the antibiotic kanamycin. Antibiotic-treated termites and control (N2-fixing) termites were exposed to different concentrations of dietary N (0, 0.21, and 0.94% N), their 15N signatures were obtained, and the percent nitrogen derived from the atmosphere within termite samples was calculated. As we hypothesized, symbiotic N2 fixation rates were negatively correlated with dietary N, suggesting that high concentrations of dietary N suppressed symbiotic N2 fixation in R. flavipes. A comparison of the 15N isotope signatures of antibiotic-treated termites with their food sources demonstrated that Δ dig = 2.284‰, and a comparison of the 15N isotope signatures of antibiotic-treated termites with control termites indicated that Δfix = -1.238‰. These are the first estimates of Δdig for R. flavipes, and the first estimate of Δfix for any N2-fixing termite species.