Ammonia can easily be assimilated into amino acids and used for silk-protein synthesis in the silkworm, Bombyx mori. To determine the metabolic pathway of ammonia assimilation, silkworm larvae were injected with methionine sulfoximine (MS), a specific inhibitor of glutamine synthetase (GS). Activity of GS in the fat body 2h after treatment with 400&mgr;g MS decreased to less than 10% of the control activity, whereas MS had no effect on the activity of glutamate dehydrogenase (GDH), another enzyme which could possibly be responsible for ammonia assimilation. Glutamine concentration in the hemolymph rapidly decreased after MS treatment, while the ammonia level in the hemolymph sharply increased. Glutamine concentration in the hemolymph 4h after injection decreased with increasing doses of MS, whereas ammonia concentration increased in proportion to the MS dose. MS strongly blocked the incorporation of (15)N label into silk-protein in larvae injected with (15)N ammonia acetate, while it slightly inhibited the incorporation of (15)N-amide glutamine into silk-protein. These results suggest that ammonia is mainly assimilated into glutamine via the action of GS and then converted into other amino acids for silk-protein synthesis and that GDH does not play a major role in ammonia assimilation in B. mori.