Summary: l-Arginine-derived nitric oxide (NO) or a labile NO-containing compound accounts for the biological activity of endothelium-derived relaxing factor (EDRF). We recently demonstrated the enzymatic conversion of l-arginine into EDRF in endothelial cells by means of a bioassay, using purified soluble guanylate cyclase as a detection system, and suggested that the EDRF-forming enzyme may be regulated by the intracellular concentration of free Ca2+. In the present article, we describe the partial purification and characterization of this enzyme from porcine cerebellum. EDRF formation was assayed by the determination of the activation of soluble guanylate cyclase; in addition, the conversion of l-arginine into l-citrulline was measured. From crude porcine cerebellum supernatant, we purified the EDRF-forming enzyme about sevenfold with 30% recovery. Guanylate cyclase was activated about 100-fold by l-arginine in the presence of the partially purified protein, which required nicotinamide adenine dinucleotide phosphate (NADPH), Ca2+, and calmodulin for enzyme activity. With regard to the cofactor concentrations producing half-maximal effects, a good correlation was found between EDRF formation and the Ca2+/calmodulin-dependent conversion of l-arginine into l-citrulline.