Glutamate synthase in tobacco cultured cells was assayed with different electron donors upon greening of etiolated cells. Ferredoxin-dependent glutamate synthase (EC 22.214.171.124) activity increased 4-fold, whereas NADH-dependent glutamate synthase (EC 126.96.36.199) and NADPH-dependent glutamate synthase (EC 188.8.131.52) activities remained constant during greening of the cells. Ferredoxin-dependent glutamate synthase and NAD(P)H-dependent glutamate synthases were distinguished by their chromatographic behavior on ferredoxin-Sepharose affinity column. The immunoglobulin G (IgG)-anti-ferredoxin-dependent glutamate synthase from rice green leaves cross-reacted with ferredoxin-dependent glutamate synthase, but NAD(P)H-dependent glutamate synthases were not immunoprecipitated with the IgG, indicating that ferredoxin-dependent glutamate synthase NAD(P)H-dependent glutamate synthase activities are carried out by distinct protein molecules in tobacco cultured cells. Some physiological functions of the enzymes are discussed.