Hypoxic pulmonary vasoconstriction (HPV) occurs with ascent to high altitude and can contribute to development of high altitude pulmonary edema (HAPE). Vascular smooth muscle contains carbonic anhydrase (CA) and acetazolamide (AZ), a CA inhibitor, blunts HPV and might be useful in the prevention of HAPE. The mechanism by which AZ impairs HPV is uncertain. Originally developed as a diuretic, AZ also has direct effects on systemic vascular smooth muscle, including modulation of pH and membrane potential; however, the effect of AZ on pulmonary arterial smooth muscle cells (PASMC) is unknown. Since HPV requires Ca²⁺ influx into PASMCs, and can be modulated by pH, we hypothesized that AZ alters hypoxia-induced changes in PASMC intracellular pH (pH_i) or Ca²⁺ concentration ([Ca²⁺]_i). Using fluorescent microscopy, we tested the effect of AZ, as well as two other potent CA inhibitors, benzolamide (BZ) and ethoxzolamide (EZ), which exhibit low and high membrane permeability, respectively, on hypoxia-induced responses in PASMCs. Hypoxia caused a significant increase in [Ca²⁺]_i, but no change in pH_i. All 3 CA inhibitors slightly decreased basal pH_i, but only AZ caused a concentration-dependent decrease in the [Ca²⁺]_i response to hypoxia. AZ had no effect on the KCl-induced increase in [Ca²⁺]_i or membrane potential. N-methyl-acetazolamide, a synthesized compound lacking the unsubstituted sulfonamide group required for CA inhibition, had no effect on pHi but also inhibited hypoxia-induced Ca²⁺ responses. These results suggest that AZ attenuates HPV by selectively inhibiting hypoxia-induced Ca²⁺ responses via a mechanism independent of CA inhibition, changes in pH_i or membrane potential.