Activity of voltage-gated K+ (KV) channels regulates membrane potential (Em) and cytosolic free Ca2+ concentration ([Ca2+]cyt). A rise in [Ca2+]cyt in pulmonary artery (PA) smooth muscle cells (SMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. Chronic hypoxia (PO2 30-35 mmHg for 60-72 h) decreased mRNA expression of Kv channel α-subunits (Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3) in PASMCs but not in mesenteric artery (MA) SMCs. Consistently, chronic hypoxia attenuated protein expression of Kv1.1, Kv1.5, and Kv2.1; reduced Kv current [I K(V)]; caused Em depolarization; and increased [Ca2+]cyt in PASMCs but negligibly affected KV channel expression, increased IK(v), and induced hyperpolarization in MASMCs. These results demonstrate that chronic hypoxia selectively downregulates Kv channel expression, reduces IK(v), and induces Em depolarization in PASMCs. The subsequent rise in [Ca2+]cyt plays a critical role in the development of pulmonary vasoconstriction and medial hypertrophy. The divergent effects of hypoxia on Kv channel α-subunit mRNA expression in PASMCs and MASMCs may result from different mechanisms involved in the regulation of KV channel gene expression.