Stimulation of soluble guanylyl cyclase in rat fetal lung fibroblasts (RFL-6 cells) was used as a sensitive assay for endothelium-derived relaxing factor/nitric oxide (EDRF/NO) formation. Intact N1E-115 cells released an EDRF/NO-like material that enhanced cyclic GMP levels in RFL-6 cells. The synthesis of this substance could be stimulated with the receptor agonist neurotensin (10 μM) or by addition of the EDRF/NO substrate L-arginine (100 μM). In Ca2+-free Locke's solution, stimulation of EDRF/NO production by both neurotensin and L-arginine was abolished. The EDRF/NO-synthesizing activity was localized in the cytosol of N1E-115 cells. The activity was lost after boiling and it was highly sensitive to Ca2+ with the major increase in activity occurring between 100 and 500 nM Ca2+. L-Arginine and NADPH were required for maximal synthesis of EDRF/NO by the enzyme(s). The synthesis of EDRF/NO was inhibited by the following antagonists of calmodulin-regulated functions (with the approximate IC50 values given in parentheses): calmidazolium (7 μM), trifluoperazine (10 μM), fendiline (80 μM), W-7 (N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide) (120 μM), and compound 48/80 (3 μg/ml). The EDRF/NO-synthesizing activity was partially purified from N1E-115 cytosol by DE 52 anion exchange chromatography. The activity was eluted with 0.1 M KCl. The enzyme(s) showed very little activity in the presence of L-arginine (100 μM) and NADPH (100 μM), but the activity could be fully restored by addition of exogenous calmodulin (EC50, ~2 units/ml). At 0.3 M KCl, a fraction eluted from the DE 52 column that was also able to fully restore the EDRF/NO-synthesizing activity. Thus, this fraction is likely to contain the endogenous Ca2+-binding protein. It is concluded that the activity of the EDRF/NO-synthesizing enzyme(s) in N1E-115 neuroblastoma cells is regulated by Ca2+ and calmodulin.