When detached soybean Glycine max (L.) Merr. cv. Hark, nodules assimilate [(13)N]N(2), the initial organic product of fixation is glutamine; glutamate becomes more highly radioactive than glutamine within 1 minute; (13)N in alanine becoms detectable at 1 minute of fixation and increases rapidly between 1 and 2 minutes. After 15 minutes of fixation, the major (13)N-labeled organic products in both detached and attached nodules are glutamate and alanine, plus, in the case of attached nodules, an unidentified substance, whereas [(13)N]glutamine comprises only a small fraction of organic (13)N, and very little (13)N is detected in asparagine. The fixation of [(13)N]N(2) into organic products was inhibited more than 99% by C(2)H(2) (10%, v/v). The results support the idea that the glutamine synthetase-glutamate synthase pathway is the primary route for assimilation of fixed nitrogen in soybean nodules.