The adaptation of contractile mechanisms of the uterine artery to pregnancy is not fully understood. The present study examined the effect of pregnancy on the uterine artery baseline Ca²⁺ sensitivity. In -escin-permeabilized arterial preparations, Ca²⁺-induced concentration-dependent contractions were significantly decreased in pregnant, as compared with nonpregnant, uterine arteries. Time course studies showed that Ca²⁺ increased phosphorylation of 20-kDa myosin light chain (MLC₂₀), which preceded the tension development in both pregnant and nonpregnant vessels. Compared with nonpregnant vessels, there was a significant increase in the protein level of MLC₂₀, and an accordance increase in the level of Ca²⁺-induced phosphorylated MCL₂₀ (MCL₂₀-P) in pregnant uterine arteries. Simultaneous measurements of MCL₂₀-P levels and contractions stimulated with Ca²⁺ in the same tissues demonstrated a significant attenuation in the ratio of tension/MLC₂₀-P in pregnant uterine arteries. Activation of PKC with phorbol 12,13-dibutrate (PDBu) potentiated Ca²⁺-induced contractions in nonpregnant but not pregnant uterine arteries. In accordance, inhibition of PKC attenuated Ca²⁺-induced contractions in nonpregnant but not pregnant uterine arteries. PDBu produced contractions in the presence or absence of Ca²⁺ in the -escin-permeabilized arteries, which was significantly decreased in pregnant, as compared with nonpregnant uterine arteries. The results suggest that pregnancy up-regulates the thick filament regulatory pathway by increasing MLC₂₀ phosphorylation, but down-regulates the thin filament regulatory pathway by decreasing the contractile sensitivity of MLC₂₀-P, resulting in attenuated baseline Ca²⁺ sensitivity in the uterine artery. In addition, PKC plays an important role in the regulation of basal Ca²⁺ sensitivity, which is downregulated during pregnancy.