Pregnancy is associated with reduced peripheral vascular resistance, underpinned by altered vascular reactivity of the endothelium and smooth muscle of key vascular beds. Failure of the maternal vasculature to adapt correctly can lead to serious complications such as preeclampsia. The peptide hormone relaxin plays a major role in regulating the maternal renal vasculature during pregnancy, but little is known about the actions of relaxin on vascular reactivity of the mesenteric artery. Therefore, this study tested the hypothesis that vascular reactivity will be compromised in the mesenteric artery of pregnant relaxin deficient (Rln-/-) mice. The vascular responses of small first order mesenteric arteries were measured in non-pregnant (oestrus) and late pregnant (day 17.5) wildtype (Rln+/+: n=5-7) and Rln-/- (n=5-7) mice using wire myography. In Rln+/+ mice, there was a significant reduction in sensitivity to the vasoconstrictor angiotensin II (AII) but not the thromboxane mimetic U46619 in late pregnant compared to non-pregnant mice. In Rln-/- mice, this normal pregnancy adaptation to AII did not occur, resulting in significantly enhanced vasoconstriction responses to AII, which were endothelium-independent. This was not a result of altered expression of AII receptors in the Rln-/- mice or an increase in reactive oxygen species as blocking with catalase, tempol and superoxide dimutase had no effect on the contractile responses to AII in either genotype. Blocking nitric oxide synthase with the inhibitor L-NAME further enhanced the vascular response to AII in both genotypes, whereas inhibition of prostanoid production with indomethacin significantly increased AII-induced contraction in day 17.5 pregnant Rln+/+ but not Rln-/- mice. In conclusion, sensitivity to AII is enhanced in the mesenteric artery of late pregnant Rln-/- mice, and is associated with a decrease in the contribution of vasodilator prostanoids but not changes in the contribution of nitric oxide, oxidative stress or expression of AII receptors.