The effects of calcitonin gene related peptide (CGRP) on constriction frequency, smooth muscle membrane potential (V_m) and endothelial V_m of guinea pig mesenteric lymphatics were examined in vitro. CGRP (1-100 nM) caused an endothelium-dependent decrease in the constriction frequency of perfused lymphatic vessels. The endothelium-dependent CGRP response was abolished by the CGRP1 receptor antagonist CGRP_(8-37) (1 µM) and pertussis toxin (100 ng.ml^-1). This action of CGRP was also blocked by the NO synthase inhibitor, N^G nitro-L-arginine (L-NOARG, 10 µM), an action that was reversed by addition of L-arginine (100 µM). cGMP, adenylate cyclase, cAMP-dependent protein kinase (PKA) and ATP-sensitive K⁺ (K⁺_ATP) channels were all implicated in the endothelium-dependent CGRP response, as it was abolished by methylene blue (20 µM), ODQ (10 µM), dideoxyadenosine (10 µM), H89 (1 µM) and glibenclamide (10 µM). CGRP (100 nM), unlike acetylcholine, did not alter endothelial [Ca²⁺]_i or V_m. CGRP (100 nM) hyperpolarized the smooth muscle V_m, an effect inhibited by L-NOARG, H89 or glibenclamide. CGRP (500 nM) also caused a decrease in constriction frequency. However, this was no longer blocked by CGRP_(8-37). CGRP (500 nM) also caused smooth muscle hyperpolarization an action that was now not blocked by L-NOARG (100 µM). It was most likely mediated by activation of the cAMP/PKA pathway and opening of K⁺_ATP channels, as it was abolished by H89 or glibenclamide. We conclude that CGRP, at low to moderate concentrations (i.e. 1 - 100 nM), decreases lymphatic constriction frequency primarily by stimulation of CGRP₁ receptors coupled to pertussis toxin (PTx) - sensitive G proteins and release of NO from the endothelium. At high concentrations (i.e. 500 nM), CGRP also directly activates the smooth muscle. Both mechanisms of activation ultimately cause PKA-mediated opening of K⁺_ATP channels and resultant hyperpolarization.