Pregnancy can influence both the resting membrane potential and the ion channel composition of the uterine myometrium. Calcium flux is essential for excitation-contraction coupling in pregnant uterus. The uterine L-type calcium channel is an important component in mediating calcium flux and is purported to play a role in parturition. This study was undertaken to characterize gestational changes in 1) the uterine contractile response to the L-type calcium channel agonist, Bay K 8644; 2) the mRNA expression of channel subunits by semiquantitative reverse transcriptase-polymerase chain reaction; and 3) estimate channel protein levels by measuring ³H-isradipine binding at the dihydropyridine binding site of the α_1c subunit utilizing saturation binding methods. Sensitivity to Bay K 8644 increases beginning at 0.8 of gestation and persists through term. The change in sensitivity is coincident with an increased mRNA expression of the α_1c and β₂ subunits but with the least detectable amounts of isradipine binding. The expressed α_1c transcript represents a novel structural variant with a 118-amino acid deletion in the III–IV linker and repeats IVS1–S3 of the protein sequence. The guinea pig uterine L-type calcium channel activity is highly regulated through gestation, but the regulation of mRNA expression may be different from regulation of protein levels, estimated by isradipine binding. The up-regulation of function, α_1c subunit mRNA expression, and isradipine binding at term gestation are consistent with a role for this ion channel in parturition.