Background: It is well known that blood pressure has a circadian rhythm in rat. However, the underlying mechanisms that modulate circadian rhythm of blood pressure have not been fully clarified. The aim of this study was to investigate the probable mechanisms that regulate time dependent variation of blood pressure. In present study, the correlations among the following s:alpha-1 adrenoceptor stimulated aortic contractions, thoracic aortic expression of Rho-kinase ll and myosin phosphatase target subunit-1 and blood biomarkers (nitric oxide, hydrogen sulfide [H2S] and total antioxidant capacity) that regulate blood pressure at six different times of the day and night were examined. Materials and Methods: Systolic blood pressure was measured every 4 h during a 24 h period in male albino Wistar rats by tail-cuff plethysmography. At each time point, contraction and relaxation responses of isolated thoracic aortas were recorded. The expression of protein from aortas was determined by western blot method. Nitric oxide, total antioxidant capacity and H2S levels were measured spectrophotometrically in plasma samples. One-way analysis of variance and student t-test was used to determine statistical differences. Results: Rat systolic blood pressure displayed a circadian rhythm, which reached the maximum at 05:00 am and minimum at 09;00 am. Diurnal variation of phenylephrine-induced contractions in the isolated thoracic aorta was also observed. Although, the Rho-kinase inhibitor Y-27632 reduced phenylephrine-induced contractions, the circadian pattern of the contractions did not change. Interestingly, Rho-kinase II and myosin phosphatase target subunit -1 protein expression in the thoracic aorta did not show significant changes throughout the day. Further, plasma levels of nitric oxide and total antioxidant capacity did not vary during the day. However, H2S levels in the systemic circulation showed circadian variation, which was the maximum at 01:00 am and minimum at 05;00 am. Conclusions: These results suggest that, in addition to alpha-1 adrenoceptor sensitivity of vessels, the circadian rhythm of plasma H2S could contribute to diurnal blood pressure variations. This highlights a potential novel experimental and therapeutic approach to blood pressure regulation.