Nitrogenase-dependent acetylene reduction activity of glasshouse-grown alfalfa (Medicago sativa L.) decreased rapidly in response both to harvesting (80% shoot removal) and applied NO(3) (-) at 40 and 80 kilograms N per hectare. Acetylene reduction activity of harvested plants grown on 0 kilogram N per hectare began to recover by day 15 as shoot regrowth became significant. In contrast, acetylene reduction activity of all plants treated with 80 kilograms NO(3) (-)-N per hectare and harvested plants treated with 40 kilograms NO(3) (-)-N per hectare remained low for the duration of the experiment. Acetylene reduction of unharvested alfalfa treated with 40 kilograms N per hectare declined to an intermediate level and appeared to recover slightly by day 15. Changes in N(2)-fixing capacity were accompanied by similar changes in levels of nodule soluble protein.After an initial lag of 24 hours, specific activities of alfalfa nodule glutamine synthetase, NADH-glutamate synthase, and NAD-glutamate dehydrogenase (oxidative amination) decreased similar to but less rapidly than acetylene reduction activity. Increased specific activities of these nodule enzymes occurred as acetylene reduction activity increased and shoot growth resumed. The observed rates of glutamine synthetase and glutamate synthase were sufficient to assimilate ammonia produced via symbiotic N(2) fixation. Nodule NADH-dependent glutamate dehydrogenase (reductive amination) specific activity was not associated with changes in acetylene reduction activity.The data indicate that host plant glutamine synthetase and NADH-glutamate synthase function to assimilate symbiotically fixed N and that NADH-dependent glutamate dehydrogenase may function in ammonia assimilation during senescence in alfalfa nodules.