Vasorelaxant effects of different atrial natriuretic peptides (ANP) were measured on rat aortic strips and mesenteric artery rings. These results were compared with the potency of the same peptides to displace125I-labelled ANP (101–126) on membrane preparations of aorta and of mesenteric vascular bed. In aortic strips and mesenteric artery rings precontracted with phenylephrine (3 × 10−8and 10−6 M, respectively), the order of potency of ANP was as follows: ANP (99–126) > ANP (101–126) > ANP (103–126) = ANP(103–125)ANP (103–123). In the displacement binding assays, the order of potency of ANP peptides was similar to that of the relaxation experiments: ANP (99–126) = ANP (101–126) > ANP (103–126) = ANP (103–125)ANP (103–123). When the vessels were precontracted by a smaller concentration of phenylephrine (10−7 M in mesenteric artery and 10−8 M in aorta), the IC50of ANP (101–126) was significantly lower than when the higher concentration of phenylephrine was used. These results show that ANP receptors in the mesenteric artery and in the aorta have similar structural requirements, according to the order of potency of different length ANP, both for binding and myotropic responses.