Stimulation of soluble guanylyl cyclase and increase in cyclic GMP in rat fetal lung fibroblasts (RFL-6 cells) was used as a bioassay to detect EDRF NO formation. The cytosolic fraction of whole rat brain synthesized an EDRF NO-like material in a process dependent on L-arginine and NADPH. The enzymatic activity was destroyed by boiling and inhibited by Nω-nitro-L-arginine. Hemoglobin and methylene blue blocked the effect of EDRF NO. When different brain regions were analyzed in the presence of L-arginine and NADPH, the cytosolic fraction from cerebellum showed the highest EDRF NO-forming activity (2-3 times higher than whole brain). Activity similar to whole brain was found in hypothalamus and midbrain. Enzymatic activities in striatum, hippocampus and cerebral cortex were about two thirds of whole brain. The lowest activity (less than half of whole brain) was found in the medulla oblongata. © 1990.