The in vitro exposure to anandamide elicits greater relaxations in mesenteric beds isolated from female compared to male rats. The present work shows that in mesenteric beds precontracted with noradrenaline the removal of endothelium increased the relaxation caused by anandamide in male and ovariectomized female but not in sham-operated female rats. The nitric oxide synthase inhibition with 100 μM Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) and the sensory in vivo denervation through neonatal administration of capsaicin also reduced anandamide-induced relaxations but these effects had the same extent in male and in female mesenteries. The content of calcitonin gene related peptide (CGRP) in mesenteric beds, that was higher in intact female than in male rats, was reduced by ovariectomy and restored to control values 21 days after a 3 weekly i.m. administration of 450 μg/kg 17β-oestradiol. This latter treatment also increased CGRP content in mesenteries from males up to the same levels observed in females. The basal release of CGRP in mesenteric beds was equivalent in either sex, but the exposure to anandamide increased CGRP release solely in female mesenteries. The ratio prostacyclin/thromboxane A2 was selectively reduced in mesenteries from male rats after exposure to anandamide, due to the decrease of the tissue levels of prostacyclin. Moreover, the cyclooxygenase-2 inhibitor 0.1 μM N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulphonamide (NS-398) diminished the relaxations caused by anandamide solely in female rats. It is proposed that relaxing factors such as CGRP and prostacyclin contribute to the higher relaxations caused by anandamide in the vasculature of female rats.