ACTH has been shown to depolarize bovine adrenal zona fasciculata cells by inhibiting a K⁺ current. The effects of this hormone on such cells have been reexamined using perforated and standard patch recording methods. In current clamp experiments, ACTH (10 nM) induced a membrane depolarization to 36 ± 1 mV (n = 56), which was mimicked by forskolin (10 µM) or by 8-(4-chlorophenylthio)-cAMP (8 mM). ACTH-induced membrane depolarizations were associated in the majority of cells with an increase in membrane conductance. In the other cells, these membrane responses could occur without change or could be correlated with a transient or with a continuous Cs⁺-sensitive decrease in membrane conductance. The depolarizations associated with an increase in membrane conductance were depressed by Cl current inhibitors diphenylamine-2-carboxylic acid (DPC; 1 mM), anthracene-9-carboxylic acid (9-AC; 1 mM), DIDS (400 µM), verapamil (100 µM), and glibenclamide (20 µM). In voltage-clamped Cs⁺-loaded cells, ACTH activated a time-independent current that displayed an outward rectification and reversed at 21.5 mV ± 2 (n = 6). This current, observed in the presence of internal EGTA (5 mM), was depressed in low Cl external solution and was inhibited by DPC, 9-AC, DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid, verapamil, and glibenclamide. ACTH-stimulated cortisol secretion was blocked by Cl channel inhibitors DIDS (400 µM) and DPC (1 mM). The present results reveal that, in addition to inhibiting a K⁺ current, ACTH activates in bovine zona fasciculata cells a Ca²⁺-insensitive, cAMP-dependent Cl current. This Cl current is involved in the ACTH-induced membrane depolarization, which seems to be a crucial step in stimulating steroidogenesis.