Lower esophageal sphincter (LES) tone depends on PGF2α and thromboxane A2acting on receptors linked to Gi3 and Gq to activate phospholipases and produce second messengers resulting in muscle contraction. We therefore examined PGF2α signal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (Eso) and LES. In Eso, PGF2α-induced contraction was inhibited by antibodies against the α-subunit of G13 and the monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and by the C3 exoenzyme of Clostridium botulinum. A [35S]GTPγS-binding assay confirmed that G13, RhoA, and ARF1 were activated by PGF2α. Contraction of Eso was reduced by propranolol, a phospholipase D (PLD) pathway inhibitor and by chelerythrine, a PKC inhibitor. In LES, PGF2α-induced contraction was inhibited by antibodies against the α-subunit of Gq and Gi3, and a [35S]GTPγS-binding assay confirmed that Gq and Gi3 were activated by PGF2α. PGF2α-induced contraction of LES was reduced by U-73122 and D609 and unaffected by propranolol. At low PGF2α concentration, contraction was blocked by chelerythrine, whereas at high concentration, contraction was blocked by chelerythrine and CGS9343B. Thus, in Eso, PGF2αactivates a PLD- and protein kinase C (PKC)-dependent pathway through G13, RhoA, and ARF1. In LES, PGF2α receptors are coupled to Gq and Gi3, activating phosphatidylinositol- and phosphatidylcholine-specific phospholipase C. At low concentrations, PGF2α activates PKC. At high concentration, it activates both a PKC- and a calmodulin-dependent pathway.