Abstract—Pulmonary artery smooth muscle cell (PASMC) proliferation contributes to increased pulmonary vascular resistance and pulmonary hypertension. Because proliferation depends on membrane potential (V_m) and because V_m is, in part, determined by Cl^- currents (I_Cl), we examined the effects of I_Cl inhibition with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on cultured rat PASMCs. DIDS (30 µmol/L) reduced cell numbers, decreased 5-bromodeoxyuridine incorporation and delayed cell cycle progression. I_Cl inhibition with 5-Nitro-2-(3-phenylpropylamino) benzoic acid (100 µmol/L) also reduced cell numbers of cultured rat PASMCs. To test the possible involvement of I_Cl in the regulation of PASMC proliferation, we measured V_m and I_Cl in both cultured (proliferating) and acutely dissociated (nonproliferating) rat PASMCs. V_m (-39.3±1.4 mV) was close to the equilibrium potential of Cl^- (-39 mV) in proliferating PASMCs but differed from equilibrium potential of Cl^- in acutely dissociated cells (-45.3±0.9 mV). DIDS and substitution of extracellular Cl^- with I^- induced V_m hyperpolarization in proliferating but not nonproliferating PASMCs. Consistent with V_m recordings, DIDS-sensitive baseline and swelling-activated (Ca²⁺-independent) I_Cls, recorded with low Ca²⁺ (<1 nmol/L) pipette solutions, were 5-fold greater in proliferating than in nonproliferating PASMCs. By contrast, Ca²⁺-activated I_Cl did not differ between proliferating and nonproliferating PASMCs. Ca²⁺-independent I_Cls were also increased in proliferating PASMCs acutely dissociated from rats exposed to hypoxia (10% O₂; 7 days). These findings are consistent with the conclusion that I_Cls regulate proliferation of PASMCs and suggest that selective I_Cl inhibition may be useful in treating pulmonary hypertension.