1. We have shown that the endocannabinoid anandamide and its stable analogue methanandamide relax rings of rabbit superior mesenteric artery through endothelium-dependent and -independent mechanisms that are unaffected by blockade of NO synthase and cyclooxygenase. 2. The endothelium-dependent component of the responses was attenuated by the gap junction inhibitor 18alpha-glycyrrhetinic acid (18alpha-GA; 50 microM), and a synthetic connexin-mimetic peptide homologous to the extracellular Gap 27 sequence of connexin 43 (43Gap 27, SRPTEKTIFII; 300 microM). By contrast, the corresponding connexin 40 peptide (40Gap 27, SRPTEKNVFIV) was inactive. 3. The cannabinoid CB1 receptor antagonist SR141716A (10 microM) also attenuated endothelium-dependent relaxations but this inhibition was not observed with the CB1 receptor antagonist LY320135 (10 microM). Furthermore, SR141716A mimicked the effects of 43Gap 27 peptide in blocking Lucifer Yellow dye transfer between coupled COS-7 cells (a monkey fibroblast cell line), whereas LY320135 was without effect, thus suggesting that the action of SR141716A was directly attributable to effects on gap junctions. 4. The endothelium-dependent component of cannabinoid-induced relaxation was also attenuated by AM404 (10 microM), an inhibitor of the high-affinity anandamide transporter, which was without effect on dye transfer. 5. Taken together, the findings suggest that cannabinoids derived from arachidonic acid gain access to the endothelial cytosol via a transporter mechanism and subsequently stimulate relaxation by promoting diffusion of an to adjacent smooth muscle cells via gap junctions. 6. Relaxations of endothelium-denuded preparations to anandamide and methanandamide were unaffected by 43Gap 27 peptide, 18alpha-GA, SR141716A, AM404 and indomethacin and their genesis remains to be established.