Abstract—Certain forms of coronary artery disease do not respond to treatment with Ca2+channel blockers, and a role for endothelin-1 (ET-1) in Ca2+antagonist-insensitive forms of coronary vasospasm has been suggested; however, the signaling mechanisms involved are unclear. We tested the hypothesis that a component of ET-1–induced coronary smooth muscle contraction is Ca2+antagonist-insensitive and involves activation of protein kinase C (PKC). Cell contraction was measured in smooth muscle cells isolated from porcine coronary artery, [Ca2+]iwas measured in fura-2 loaded cells, and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific PKC antibodies using Western blot analysis. In Hank’s solution (1 mmol/L Ca2+), ET-1 (10−7mol/L) caused a transient increase in [Ca2+]i(236±14 nmol/L) followed by a maintained increase in [Ca2+]i(184±8 nmol/L) and 35% cell contraction. The Ca2+channel blockers verapamil and diltiazem (10−6mol/L) abolished the maintained ET-1–induced [Ca2+]i, but only partially inhibited ET-1–induced cell contraction to 18%. The verapamil-insensitive component of ET-1 contraction was inhibited by the PKC inhibitors calphostin C and &egr;-PKCV1–2. ET-1 caused translocation of Ca2+-dependent &agr;-PKC and Ca2+-independent &egr;-PKC from the cytosolic to the particulate fraction that was inhibited by calphostin C. Verapamil abolished ET-1–induced translocation of &agr;-PKC, but not that of &egr;-PKC. Phorbol 12-myristate 13-acetate (10−6mol/L), a direct activator of PKC, caused 22% cell contraction, with no increase in [Ca2+]i, and translocation of &egr;-PKC that was inhibited by calphostin C, but not by verapamil. KCl (51 mmol/L), which stimulates Ca2+influx, caused 35% cell contraction and increase in [Ca2+]i(291±11 nmol/L) that were inhibited by verapamil, but not by calphostin C, and did not cause translocation of &agr;- or &egr;-PKC. In Ca2+-free (2 mmol/L EGTA) Hank’s solution, ET-1 caused 15% cell contraction, with no increase in [Ca2+]i, and translocation of &egr;-PKC that were inhibited by &egr;-PKC V1–2 inhibitory peptide. Thus, a significant component of ET-1–induced contraction of coronary smooth muscle is Ca2+antagonist-insensitive and involves activation and translocation of Ca2+-independent &egr;-PKC, and may represent a signaling mechanism of Ca2+antagonist-resistant forms of coronary vasospasm.