The electrical properties of the apical membrane of isolated rabbit parietal cells were studied using the patch-clamp technique. The apical membrane of the parietal cells plated on Matrigel and maintained in culture conditions was identified by lectin-binding studies. Cell-attached and excised inside-out patches from 10-4 M cimetidine-treated parietal cells infrequently contained Cl- channels (9% of the patches). A single class of voltage-dependent outwardly rectifying Cl- channels with 24 ± 1-pS conductance was observed in 75% of the patches from cells stimulated (acid secreting) by 10-4 M histamine. Other anions passed through these channels with a permeability sequence of I- (1.2) > Br- (1.1) ≥ Cl- (1.0) > NO3- (0.7) > SO42- (0.1), but there was a very low permeability for Na+ or K+ (P(Cl-)/P(Na+) or P(Cl-)/P(K+) > 5). In inside-out patch configurations the Cl- channel was insensitive to Ba2+ and stilbene derivatives but was inhibited by diphenylamine-2-carboxylic acid in a manner characteristic of a reversible open-channel blocker. It is concluded that H2-receptor agonist stimulation of acid secretion by rabbit parietal cells activates Cl- channels channels in the apical cell membrane.