C-type natriuretic peptide (CNP) is an endothelium-derived factor which causes vascular smooth muscle relaxation. The mechanism by which CNP acts is controversial. Most reports indicate that CNP acts on NPR-B receptors to increase smooth muscle cyclic GMP levels. However, it has recently been proposed that CNP instead stimulates NPR-C receptors to activate inwardly rectifying K⁺ channels, and that CNP represents a major endothelium dependent hyperpolarizing factor released by acetylcholine in rat mesenteric resistance arteries. We therefore examined the properties and mechanism of the response to CNP in rat mesenteric resistance arteries (RMA), superior mesenteric arteries (SMA) and isolated coronary arteries. CNP (1-1000nM) failed to induce a relaxation in the RMA and coronary arteries but caused a substantial transient relaxation in the SMA (1µM: 77±3% relaxation, n=6). This relaxation was insensitive to the combination of TRAM-34 (1μM) and apamin (100nM) (63±5% relaxation, n=6) and pre-treatment with pertussis toxin (400ng/ml, 77±4% relaxation, n=6) but was antagonized by 25mM K⁺ (-3±3% relaxation, n=6, P<0.001), and the combination of Ba²⁺ (10µM) and ouabain (100µM) (14±3% relaxation, n=6, P<0.01). Relaxation was also prevented by the BK_Ca channel inhibitor iberiotoxin (100nM, -10±3% relaxation, n=6, P<0.001), which however had no effect on acetylcholine-induced relaxations. The NPR-C agonist cANF (1μM) had no effect in either the RMA or SMA (0±1%, n=6), suggesting that CNP was acting through the NPR-B receptor in the SMA. Relaxation was insensitive to the protein kinase G inhibitor KT5823 (2μM, 78±3% relaxation, n=6) but was inhibited by milrinone (30μM, 19±5% relaxation, n=6, P<0.01), a drug which selectively inhibits cyclic GMP-dependent phosphodiesterase, (PDE3) and by H-89 (30μM, 2±2% relaxation, n=5, P<0.01), which inhibits protein kinase. Relaxation was also abolished by the phospholipase C antagonist U73122 (5μM, 11±1% relaxation, n=4, P<0.001) and by [Ca²⁺]_i store-releasing agent caffeine (0.5mM, 6±3% relaxation, n=6, P<0.001)). Relaxation of the SMA to 8-Br-cyclic GMP (200μM, 83±4% relaxation, n=6) was also inhibited by U73122 and Milrinone (5±5% relaxation, n=5, P<0.01), but not KT5823. The results indicate that CNP relaxes the SMA via the NPR-B receptor, which would be expected to raise levels of cyclic GMP. We propose that then inhibits PDE3, leading to a rise in intracellular cyclic AMP levels and the accumulation of Ca²⁺ in the sarcoplasmic reticulum. This may result in an increase in vectorial Ca²⁺ release and the opening of BK_Ca channels, leading to membrane hyperpolarization and relaxation. The results also demonstrate that CNP is unlikely to be an EDHF in the resistance vasculature, at least in the rat.