We have recently proposed that an endogenous cannabinoid may be an endothelium-derived hyperpolarizing factor (EDHF), and we have now characterized the cannabinoid receptors mediating these responses. EDHF-mediated vasorelaxations to carbachol (ED50=3.26+/-0.57 nmol; the maximum relaxation, Rmax = 87.0+/-2.5%) were opposed by the selective cannabinoid CB1 antagonist, LY320135: at 2 microM ED50 for carbachol was 10.4+/-2.6 nmol and Rmax was 66.9+/-6.2%, at 10 microM ED50 was 15.9+/-4.0 nmol and Rmax was 34.0+/-4.3%. However, these responses were unaffected by another putative CB1 ligand, AM630 (10 microM), or a CB2 selective antagonist, SR 144528 (100 nM-1 microM). None of the antagonists influenced vasorelaxation to either the potassium channel activator levcromakalim or sodium nitroprusside. Coupled to our previous observation that the CB1 receptor antagonist SR141716A opposes EDHF-mediated relaxation, the present observations point to the involvement of a cannabinoid receptor, which may be CB or CB1-like, in EDHF-mediated vasorelaxation.