Previously, we have demonstrated that ERK/PKC signaling pathways play a key role in the regulation of Ca²⁺ sensitivity and contractility of the uterine artery. The present study tested the hypothesis that ERK and PKC differentially regulate myosin light chain phosphatase (MLCP) activity by phosphorylation of the MLCP regulatory subunit MYPT1 and CPI-17. Agonist-induced contractions and phosphorylation of MYPT1/Thr⁶⁹⁶, MYPT1/Thr⁸⁵⁰, and CPI-17/Thr³⁸ were measured simultaneously in the same tissues of isolated near-term pregnant ovine uterine arteries. Phenylephrine produced time-dependent concurrent increases in the phosphorylation of ERK_44/42 and MYPT1/Thr⁸⁵⁰, which preceded contractions. In addition, phenylephrine induced phosphorylation of CPI-17/Thr³⁸ that was concurrent with the contractions. In contrast, phenylephrine did not induce phosphorylation of MYPT1/Thr⁶⁹⁶. PD098059 inhibited phosphorylation of ERK_44/42 and the initial peak phosphorylation of MYPT1/Thr⁸⁵⁰, but did not affect CPI-17/Thr³⁸ phosphorylation. PKC activation by PDBu induced a time-dependent phosphorylation of CPI-17/Thr³⁸ that preceded the contractions of the uterine artery. In addition, PDBu activated PKC, and induced co-immunoprecipitation of PKC with caldesmon. The results suggest that phosphorylation of MYPT1/Thr⁸⁵⁰ and CPI-17/Thr³⁸ play important roles in the regulation of agonist-mediated Ca²⁺ sensitivity in the uterine artery, which are regulated in part by ERK and PKC, respectively. In addition, phosphorylated CPI-17 may regulate the Ca²⁺ sensitivity through the thin filament regulatory pathway in the uterine artery.