We characterized bradykinin (BK)-induced changes in the intracellular Ca²⁺ concentration ([Ca²⁺]i) and membrane potential in cultured rat myenteric neurons using ratiometric Ca²⁺ imaging with fura-2 and the whole-cell patch-clamp technique, respectively. BK evoked a dose-dependent increase of [Ca²⁺]i that was abolished by HOE 140, a B2 receptor antagonist but not by [Lys-des-Arg⁹]-BK, a B1 receptor antagonist. [Lys-des-Arg⁹]-HOE140, a B1 receptor agonist, failed to cause a [Ca²⁺]i response. Double staining with antibodies against the B2 receptor together with PGP9.5 or S100 indicated that B2 receptors were expressed in neurons and glial cells. The BK-evoked [Ca²⁺]i increase was suppressed by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and potentiated by prostaglandin E₂ (PGE₂). The release of PGE₂ from cultured myenteric plexus cells was increased by BK. BK induced a large increase in [Ca²⁺]i in neurons when myenteric plexus cells were cultured at the high density but not at the low density, and caused a small increase in [Ca²⁺]i in neurons when proliferation of enteric glial cells was suppressed. BK evoked a slow and sustained depolarization in myenteric neurons, which was sensitive to indomethacin. These results indicated that BK caused a [Ca²⁺]i increase and depolarization in rat myenteric neurons through the activation of B2 receptors, which was partly associated with PGE₂ released from glial cells in response to BK. It is suggested that a neuron–glial interaction plays an important role in the effect of BK in the rat myenteric plexus.