Ca(2+)-activated Cl- current in freshly isolated smooth muscle cells from rat portal vein was studied using the whole-cell patch-clamp technique. Simultaneously, the free-cytosolic Ca2+ concentration (Cai) was estimated using emission from the dye Indo-1. Pretreatment of the cells with amytal and carbonyl-cyanide-m-chlorophenylhydrazone, which reduced the intracellular adenosine triphosphate concentration, was used to weaken the cellular Ca2+ homeostatic system. Cai of treated cells slowly increased during perfusion with an external Ca(2+)-containing solution. This rise in Cai gradually activated a Ca(2+)-dependent Cl- current which allowed the study of the relationship between activation of this current and Cai levels. The threshold Cai for activation of Cl- channels was around 180 nM and full activation occurred at 600 nM. The Cai dependence of the Cl- channels was not changed during application of noradrenaline and did not depend on the membrane potential. The gating of Ca(2+)-dependent Cl- channels of rat portal vein myocytes seems to be mainly controlled by intracellular Ca2+.