Protein kinase C (PKC) plays an important role in the regulation of uterine artery contractility and its adaptation to pregnancy. The present study tested the hypothesis that PKC differentially regulates ₁-adrenoceptor-mediated contractions of nonpregnant and pregnant uterine arteries. Phenylephrine-induced contractions of uterine arteries isolated from nonpregnant (NPUA) and near-term pregnant (PUA) sheep were determined in the absence or presence of PKC activator, phorbol 12, 13-dibutyrate (PDBu). In NPUA, PDBu produced a concentration-dependent potentiation of phenylephrine-induced contractions and shifted the dose-response curve to the left. In contrast, in PUA PDBu significantly inhibited phenylephrine-induced contractions and decreased their maximum response. Simultaneous measurement of contractions and intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) in the same tissues revealed that PDBu inhibited phenylephrine-induced [Ca²⁺]_i and contractions in PUA. In NPUA, PDBu increased phenylephrine-induced contractions without changing [Ca²⁺]_i . Western blot analysis showed six PKC isozymes, , _I, _II, , , and in uterine arteries, among which _I, _II and isozymes were significantly increased in PUA. In contrast, PKC was decreased in PUA. In addition, analysis of sub-cellular distribution revealed a significant decrease in the particulate/cytosolic ratio of PKC in PUA, as compared with NPUA. The results suggest that pregnancy induces a reversal of PKC regulatory role on ₁-adrenoceptor-mediated contractions from a potentiation in NPUA to an inhibition in PUA. The differential expression of PKC isozymes and their sub-cellular distribution in uterine arteries appears to play an important role in the regulation of Ca²⁺ mobilization and Ca²⁺ sensitivity in 1-adrenoceptor-mediated contractions and their adaptation to pregnancy.