Combined treatment of isolated rat gastric mucosal (RGM1) cells with interleukin (IL)-1beta and transforming growth factor (TGF)-alpha resulted in expression of inducible nitric oxide synthase (iNOS) mRNA and iNOS protein 24 hr after the treatment. Constitutive NOS (nNOS) protein was not proved in the cells and not activated by IL-1beta+TGFalpha. Although IL-1beta and TGF-alpha alone exerted little or no effect on NO2 production, their combination gradually increased NO2- production from 12 to 24 hr following treatment. NO2- production stimulated by IL-1beta + TGFalpha was significantly reduced by N(G)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, yet not by D-NAME. S-nitroso-N-acetyl-D,L-penicillamine and sodium nitropruside significantly inhibited both spontaneous and TGF-alpha stimulated DNA synthesis. Nonetheless, L-NAME did not affect the inhibition by IL-1beta of TGF-alpha-stimulated proliferation of RGM1 cells, eliminating the possibility of involvement of NO in the underlying mechanisms.