Urocortin, a vasodilatory peptide related to corticotropin-releasing factor, may be an endogenous regulator of blood pressure. In vitro, rat tail arteries are relaxed by urocortin by a cAMP-mediated decrease in myofilament Ca2+ sensitivity through a still unclear mechanism. Here we show that contraction of intact mouse tail arteries induced with 42 mmol/L KCl or 0.5 micromol/L noradrenaline was associated with a approximately 2-fold increase in the phosphorylation of the regulatory subunit of myosin phosphatase (SMPP-1M), MYPT1, at Thr696, which was reversed in arteries relaxed with urocortin. Submaximally (pCa 6.1) contracted mouse tail arteries permeabilized with alpha-toxin were relaxed with urocortin by 39+/-3% at constant [Ca2+], which was associated with a decrease in myosin light chain (MLC20Ser19), MYPT1Thr696, and MYPT1Thr850 phosphorylation by 60%, 28%, and 52%, respectively. The Rho-associated kinase (ROK) inhibitor Y-27632 decreased MYPT1 phosphorylation by a similar extent. Inhibition of PP-2A with 3 nmol/L okadaic acid had no effect on MYPT1 phosphorylation, whereas inhibition of PP-1 with 3 micromol/L okadaic acid prevented dephosphorylation. Urocortin increased the rate of dephosphorylation of MLC20Ser19 approximately 2.2-fold but had no effect on the rate of contraction under conditions of, respectively, inhibited kinase and phosphatase activities. The effect of urocortin on MLC20Ser19 and MYPT1 phosphorylation was blocked by Rp-8-CPT-cAMPS and mimicked by Sp-5,6-DCl-cBIMPS. In summary, these results provide evidence that Ca(2+)-independent relaxation by urocortin can be attributed to a cAMP-mediated increased activity of SMPP-1M which at least in part is attributable to a decrease in the inhibitory phosphorylation of MYPT1.