Methods have been developed for identifying the pathway of assimilation of N2-derived nitrogen. The products of fixation of 13N-labeled nitrogen gas ([13N]N2), and the distribution of 13N within glutamine, were determined after short periods of labeling (approximately 1 to 120 s) and also in pulse-chase experiments. Ammonia, the amide nitrogen of glutamine, and the alpha-amino nitrogen of glutamate, in that order, were the first observed products of fixation of N2 by the cyanobacterium (blue-green alga), Anabaena cylindrica. This sequence of the formation of nitrogenous products was confirmed by the use of inhibitors. The presence of 1 mM methionine sulfoximine permitted continued formation of 13NH3, while virtually preventing 13N-labeling of amino acids. In the presence of 1 mM azaserine, glutamine was labeled, but not other amino acids. Our observations demonstrate unequivocally that N2-derived nitrogen fixed by this organism is metabolized initially by the glutamine synthetase/glutamate synthase pathway.