Endothelium-derived relaxing factor (EDRF) released from cultured endothelial cells was assayed by examining changes in cyclic GMP levels of rat lung fibroblasts. N omega-nitro-L-arginine and NG-monomethyl-L-arginine inhibited basal and A23187-, ATP- and melittin-induced EDRF release, and the inhibition was prevented with L-arginine. The IC50 values of N omega-nitro-L-arginine and NG-monomethyl-L-arginine for EDRF release evoked with 1 microM A23187 were 230 nM and 16 microM, respectively. N omega-nitro-L-arginine and NG-monomethyl-L-arginine did not affect cyclic GMP accumulation in the fibroblasts with atrial natriuretic factor or sodium nitroprusside. Thus, N omega-nitro-L-arginine is 70 times more potent than NG-monomethyl-L-arginine as a specific inhibitor of EDRF formation/release.