Chloride (Cl-) channels were characterized in vascular smooth muscle cells (VSMC) using radioisotope flux and patch-clamp electrophysiological techniques. Transmembrane 125iodine (125l) efflux from subcultured (Passage 1-5) rat aortic VSMCs was used as an indicator of Cl- movements to study the relationship between intracellular calcium concentration ([Ca2+]1) and CI- channel activity. Angiotensin II (Ang II) (10-7M) and adenosine 5'-triphosphate (ATP) (10-4M) induced rapid increases (9,7- and 14.9-fold, respectively) in 125I efflux rates. We found that both Ang II-and ATP-stimulated 125I efflux and [Ca2+]I increases were completely abolished after brief incubation (20 μM, 20 min) with the acetoxymethyl ester of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), a membrane-permeable Ca2+ chelator. However, when external EGTA was used to blunt agonist-stimulated Ca2+ influx, 125l efflux was still increased in response to Ang II and ATP. These data suggest that Ca2+ release from intracellular sites is sufficient to activate CI- channels in response to Ang II and ATP. Using standard patch-clamp electrophysiological techniques, we found that Ang II, a Ca2+-mobilizing agonist, stimulated outward Cl- currents (gcl = 75 pS) in cell-attached (C/A) patches of primary and subcultured VSMCs. Collectively, these data suggest that Ang II and other vasoconstrictor agents stimulate Cl- channel activity via increases in [Ca2+]l. Cl- channel activation may help to depolarize the VSMC membrane leading to increased Ca2+ influx during agonist stimulation. © 1995, SAGE Publications. All rights reserved.