Studies in vitro have demonstrated that vasoconstrictor agents increase intracellular Ca²⁺ and activate protein kinase C (PKC) to elevate vascular tone. The aim of the present study was to determine the importance of these signaling pathways for angiotensin II (ANG II) and thromboxane A₂ (TxA₂) in regulating mesenteric blood flow (MBF) in vivo. In anesthetized rats increasing doses of ANG II or the TxA₂ agonist U-46619 were administered into the superior mesenteric artery to reduce MBF. Intra-arterial infusion of inhibitors served to examine the contribution of different pathways: 8-(diethylamino)octyl 3,4,5-trimethoxybenoate hydrochloride (TMB-8) to inhibit intracellular Ca²⁺ release, nifedipine to block transmembrane Ca²⁺ influx through the L-type Ca²⁺ channel, and staurosporine to inhibit PKC. Each of the inhibitors attenuated ANG II-induced reductions in MBF, and all dose-response curves were shifted to the right to an approximately threefold higher ANG II dose. Combinations of the inhibitors revealed that their effects were additive; together they abolished the vasoconstrictor action of ANG II completely. In contrast, the dose-response curve for U-46619 was not affected by any of the inhibitors infused either separately or together. The results demonstrate that a rise in intracellular Ca²⁺ and activation of PKC are major mediators of the vasoconstrictor effect of ANG II in mesenteric circulation, but they play a subordinate role, if any, for the effects of TxA₂. Because TxA₂ plays a major role only under pathological conditions, the uncontrolled vasoconstriction appears to be associated with the recruitment of novel signal transduction pathways.