Thromboxane A2 (TXA2), a platelet derived short lived derivative of arachidonic acid, is known to be involved in autoregulation of the brain's circulatory system under both physiological and pathophysiological conditions. TXA2-receptor activation in vascular smooth muscles by the stable TXA2 mimetic, U-46619 predominantly activates the RhoA/Rho-kinase Ca²⁺-sensitization pathway resulting in inhibition of the myosin light chain phosphatase (MLCP) activity and, hence, a Ca²⁺-independent increase in MLC₂₀ phosphorylation and contractile force. The U-46619 induced inhibition of MLCP activity is known to be mediated by an increase in the inhibitory phosphorylation of the MLCP targeting subunit (MYPT1) at either T696 or T853. Surprisingly, in rat cerebral vasculature, activation of the TXA2-receptor by U-46619 also resulted in increased phosphorylation of MYPT1 at Ser695, a site known to be phosphorylated by both cAMP/cGMP dependent protein kinases leading to Ca²⁺-desensitization. Inhibition of nitric oxide signaling completely abrogated basal MYPT1-S695 phosphorylation, and significantly increased, and potentiated U-46619 induced MYPT1-T853 and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of Ca²⁺-sensitization. Surprisingly, a fall in S695 phosphorylation did not result in an increase in phosphorylation of the T696 site. cGMP treatment inhibited U-46619 induced force and MLC20 phosphorylation but did not decrease T696 or T853 phosphorylation, indicating that the cGKI vasorelaxant effects are not through dephosphorylation of MYPT1. In contrast, in pulmonary arteries, basal MYPT1-S695 phosphorylation is 40% lower than in cerebral vessels and is abolished by mechanical removal of the endothelium. However, U-46619 treatment of pulmonary arteries does not result in an increase in MYPT1-S695 phosphorylation. In conclusion, our data suggests that the basal level of arterial smooth muscle MYPT1-S695 phosphorylation is under the control of the endothelium. Interestingly, TXA2 induces a bi-directional regulation of cerebral arterial tone in which both RhoA/Rho-kinase mediated Ca²⁺ sensitization and the NO/cGMP/cGKI Ca²⁺-desensitization pathways are activated.