NITRIC oxide is the major endothelium-derived relaxing factor (EDRF)(1-3), and it is thought to relax smooth muscle cells by stimulation of guanylate cyclase, accumulation of its product cyclic GMP, and cGMP-dependent modification of several intracellular processes(4-5), including activation of potassium channels through cGMP-dependent protein kinase(6,7). Here we present evidence that both exogenous nitric oxide and native EDRF can directly activate single Ca2+-dependent K+ channels (K-Ca(+)) in cell-free membrane patches without requiring cGMP. Under conditions when guanylate cyclase was inhibited by methylene blue, considerable relaxation of rabbit aorta to nitric oxide persisted which was blocked by charybdotoxin, a specific inhibitor of K-Ca(+) channels. These studies demonstrate a novel direct action of nitric oxide on K-Ca(+) channels.