The circadian rhythm in blood pressure is characterized by a nocturnal “dip” in man, coupled to a decrease in peripheral resistance (Sindrup et al., 1991). Peripheral resistance reflects vascular tone set by the contractile status of vascular smooth muscle (VSM), which is modulated by endothelial production of nitric oxide (NO). Circadian variation in endothelial-dependent relaxation of VSM is suggested from experiments showing that the acetylcholine-induced increase in forearm blood flow in man peaks at 0800 with a trough at 2000 hours, and this difference was reduced by L-NAME (Shaw et al., 2001). We have therefore looked at the impact of the time-of-day on contractile response of mesenteric resistance vessels to phenylephrine and its modulation by acetylcholine. Adult male Wistar rats were housed with a 12 hour light/dark cycle and euthanized by stunning and cervical dislocation, three hours into the animals resting (light) or active (dark) period. The mesentery was removed and recordings of contractile force were made from 2-4 mm ring segments of the superior mesenteric artery first-order branches, using a wire-myograph at 37°C. (Data are mean ± S.E.M; n=animals/preparations; compared with t-test or 2-way ANOVA). Dose-response curves of contraction strength to phenylephrine (PHE) show a time-of-day variation in the maximal amplitude of contraction (200μM), which peaked during the resting period at 18.6 ± 1.2 mN (n=5/21) versus 11.8 ± 0.9 mN in the active period (n=4/28; p<0.001), with no difference in EC50 at 3.3±0.4 versus 4.1±0.7 μM. Inhibition of endothelial nitric oxide synthase (eNOS) with L-NAME (400μM) resulted in a further increase in the maximal PHE-induced contraction, which was significantly greater in active than resting period arteries at 19.5 ± 1.4 mN (n=4/24) in resting versus 17.7 ± 0.8 mN in active period (n=5/31; p<0.01). Dose-response curves of the relaxation of the PHE-induced contraction by acetylcholine exhibit strong time-of-day variation, with the dose-response curve shifted to the left during the active period with an EC50 of 232 ± 31 nM (n=5/22) during the resting and 58.6 ± 11 nM (n=4/22; p<0.001) in the active period. qRT-PCR analysis of mRNA and western blot measurement of protein levels of eNOS in snap frozen vessels show a 3.5-fold increase in eNOS gene expression during the animal’s active period, which translated to 2.4-fold increase (p<0.05; n=10) in total eNOS protein and 1.5-fold increase of phosphorylated-S1177 eNOS p<0.01; n=10). In endothelium denuded vessels, the time-of-day variation in contractile response to PHE was absent. Our data show the presence of a time-of-day variation in excitation-contraction coupling of VSM, which is endothelial-dependent and appears to involve NO-signalling, This has implications for both vascular contractility and clot formation.