It has been proposed that a hypoxia-induced inhibition of Na⁺-Ca²⁺ exchange (NCX), contributes to hypoxic pulmonary vasoconstriction (HPV). By recording isometric tension development in rat intrapulmonary arteries (IPA), we examined the effect on HPV of maneuvers which reduce the ability of NCX to regulate [Ca²⁺]_i. In some experiments Fura PE-3 was also used to monitor [Ca²⁺]_i. HPV was elicited in IPA which were pretreated with 10µM diltiazem and slightly preconstricted with PGF2, which enhances the hypoxic response. Substitution of Na⁺ with Li⁺ increased HPV and the associated rise in [Ca²⁺]_i. Pretreatment with ouabain (100µM) to diminish the Na⁺ gradient or with the reverse-mode NCX inhibitor KB-R7943 (3 or 10µM) had no significant effect on HPV. Combined treatment with ouabain and low-Na⁺ (24 mM) solution enhanced HPV strongly. The role of NCX in Ca²⁺ extrusion was examined by assessing the decrease in [Ca²⁺]_i in Ca²⁺-free PSS either containing or lacking [Na⁺] following a high K⁺- induced loading of cellular [Ca²⁺]. Although the large initial rapid fall in [Ca²⁺]_i was Na⁺-independent, final recovery of [Ca²⁺]_i to its basal level was delayed in the absence of Na⁺. Therefore, HPV persisted or was increased under conditions in which forward mode NCX was already attenuated or prevented, demonstrating that inhibition of NCX by hypoxia is unlikely to initiate HPV. Instead, NCX appears to act to inhibit HPV as would be expected if it is functioning to extrude Ca²⁺.