The fate of the interstitial atom of the nitrogenase cofactor during substrate turnover has remained a topic of interest since the discovery of this atom more than a decade ago. In this study, we labeled the interstitial carbide atom with C-14 and C-13 isotopes and traced the fate of the isotope under turnover conditions. Our results show that the interstitial carbide cannot be exchanged upon turnover, nor can it be used as a substrate and incorporated into the products. These observations point to a role of the interstitial carbide in stabilizing the cofactor structure, although a function of this atom in indirectly modulating the reactivity of the cofactor or directly interacting with the substrate cannot be excluded.