The effects of the Cl− channel antagonists, niflumic acid (NFA), dichloro-diphenylamine 2-carboxylic acid (DCDPC) and diisothiocyanato-stilbene-2,2′-disulphonic acid (DIDS) on Ca2+-activated Cl− current (ICl(Ca)) evoked by adding fixed intracellular calcium concentrations ([Ca2+]i) to the pipette solution were studied in rabbit pulmonary artery myocytes. With 250 and 500 nm[Ca2+]i bath application of NFA (100 μm) increased inward current at negative potentials, but inhibited outward current at positive potentials. On wash out of NFA, ICl(Ca) was greatly enhanced at all potentials. When external Na+ ions were replaced by N-methyl-d-glucamine (NMDG+) NFA still enhanced ICl(Ca) at negative potentials but the increase of ICl(Ca) on wash out was blocked. When the mean reversal potential (Er) of ICl(Ca) was shifted to negative potentials by replacing external Cl− with SCN−, NFA increased inward current but blocked outward current suggesting that the effect of NFA is dependent on current flow. Inclusion of NFA in the pipette solution had no effect on ICl(Ca). Voltage jump experiments indicated that ICl(Ca) displayed characteristic outward current relaxations at +70 mV and inward current relaxations at −80 mV that were abolished by NFA. DCDPC (100 μm) produced similar effects to NFA but 1 mm DIDS produced inhibition of ICl(Ca) at both positive and negative potentials and there was no increase in current on wash out of DIDS. These results suggest that NFA and DCDPC, but not DIDS, simultaneously enhance and block ICl(Ca) by binding to an external site, probably close to the mouth of the chloride channel.