The possible contribution of Ca²⁺-activated Cl^- channel (ICl(Ca)) and myosin light chain kinase (MLCK) to non-adrenergic, non-cholinergic slow IJP (sIJP) were studied using conventional intracellular microelectrode recordings in circular smooth muscle of opossum esophageal body and guinea-pig ileum perfused with Kreb's solution containing atropine (3 µM), guanethidine (3 µM) and substance P (1 µM). In opossum esophageal circular smooth muscle, resting membrane potential (MP) was -51.9 ± 0.7 mV (n = 89) with MP fluctuations of 1 - 3 mV. A single square wave nerve stimulation of 0.5 ms duration and 80 V induced a sIJP with amplitude of 6.3 ± 0.2 mV, half amplitude duration of 635 ± 19 ms and rebound depolarization amplitude of 2.4 ± 0.1 mV (n = 89). 9-anthroic acid (A-9-C), niflumic acid (NFA), wortmannin and 1-(5-chloronaphthalene-1-sulfonyl)-1-H-hexahydro-1,4-diazepine (ML-9) abolished MP fluctuations, sIJP and rebound depolarization in a concentration-dependent manner. A-9-C and NFA, but not wortmannin and ML-9, hyperpolarized MP. In guinea-pig ileal circular smooth muscle, nerve stimulation elicited an IJP composed of both fast (fIJP) and slow components (sIJP), followed by rebound depolarization. NFA (200 µM) abolished sIJP and rebound depolarization but left the fIJP intact. These data suggest that in the tissues studied, activation of I_Cl(Ca), which requires MLCK, contributes to resting MP, and that closing of I_Cl(Ca) is responsible for sIJP.