Ca(2+)-activated Cl- currents were studied in isolated cells from rat portal vein smooth muscle in short-term primary culture using the whole-cell patch-clamp technique. Cl- currents can be activated separately by Ca2+ release from intracellular stores (in response to external applications of caffeine or noradrenaline) and by Ca2+ influx through voltage-dependent Ca2+ channels. The effects of several Cl- channel blockers and of spironolactone (a substance known to reduce internal Ca2+ loading) on both Cl- and Ca2+ currents were examined. Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and diphenylamine-2,2'-dicarboxylic acid (DPC) inhibited the Ca(2+)-activated Cl- current (IC50 values between 16.5 and 306 microM) with no effects on the inward Ca2+ current and on internal Ca2+ loading (testing by measuring the Ca(2+)-activated K+ current). These results indicate that the inhibition of Cl- current by these compounds is due to a direct interaction with the Cl- channel. In contrast, spironolactone inhibited both K+ and Cl- currents (IC50 = 7.6 microM) by reducing the amount of Ca2+ located in the internal stores, whereas the Cl- current activated by Ca2+ current through T-type Ca2+ channels was unchanged. This preparation and the protocols developed in this study appears to be appropriate for analysis of substances interfering with Cl- channels or intracellular Ca2+ stores.