Regulation of nitrogen fixation by ammonium and glutamate was examined in Rhizobium sp. 32H1 growing in defined liquid media. Whereas nitrogenase synthesis in Klebsiella pneunoniae is normally completely repressed during growth on NH4+, nitrogenase activity was detected in cultures of Rhizobium sp. grown with excess NH4+. However, an "ammonium effect" on activity was invariably observed in cultures grown on NH4+ as sole nitrogen source; the nitrogenase activity was, depending on conditions, 14 to 36% of that of comparable glutamate-grown cultures. Glutamate inhibited utilization of exogenous NH4+ and, in one of two procedures described, glutamate partially alleviated the ammonium effect on nitrogenase activity. NH4+, apparently produced from N2, was excreted into the culture medium when growth was initiated on glutamate, but not when NH4+ was thesole source of fixed nitrogen for growth. These findings are discussed in relation to nitrogen fixation by Rhizobium bacteroids.