Guinea-pig mesenteric lymphatic vessels exhibit vasomotion through a pacemaker mechanism involving intracellular Ca²⁺ release and resultant spontaneous transient depolarizations (STDs) of the smooth muscle membrane potential. This study presents a detailed characterization of the effects of cyclopiazonic acid (CPA) on this pacemaker activity. Microelectrode recordings from smooth muscle in vessel segments revealed that application of CPA (1-10 µM) caused a hyperpolarization, accompanied by a decrease in the frequency and amplitude of STDs. The CPA-induced hyperpolarization was abolished after destruction of the endothelium and in the presence of N^G-nitro-L-arginine (L-NOARG, 100 µM) or ODQ (10 µM), suggesting a contribution of endothelium-derived nitric oxide (EDNO) in this response. In the absence of EDNO-induced effect, CPA decreased the frequency and amplitude of STDs recorded before and in the presence of the thromboxane A₂ mimetic U46619, norepinephrine or thimerosal. CPA abolished U46619-induced vasomotion as determined by measuring constriction-associated [Ca²⁺]_i using the ratiometric Ca²⁺ indicator fura-2. The endothelial actions of CPA were compared to those of acetylcholine (ACh), known to cause EDNO release in this preparation. While CPA and ACh both increased endothelial [Ca²⁺]_i and depolarized the membrane potential, the kinetics of action for both parameters were markedly slower for CPA than ACh. These results suggest that CPA first hyperpolarizes the lymphatic smooth muscle and decreases STD frequency and amplitude through endothelial release of EDNO and second, consistent with the action of CPA to inhibit sarcoplasmic reticulum Ca²⁺-ATPase and to deplete Ca²⁺stores, it further reduces STD activity. Inhibition of the lymphatic smooth muscle pacemaker mechanism is thought to abolish agonist-induced vasomotion.