Neonatal persistent pulmonary hypertension (PPHN), caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane:prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O₂ for 3 days; control myocytes from normoxic piglets were cultured in 21% O₂. PPHN was induced in newborn piglets by 3 day hypoxic exposure (FiO₂ 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca²⁺ mobilization to thromboxane mimetic U46619 and ATP was quantified using fura-2AM. 3 day hypoxic exposure in vitro results in increased basal [Ca²⁺]i, faster and heightened peak Ca²⁺ response, and decreased U46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O₂. Paradoxically, cell surface thromboxane receptor abundance is decreased due to internal translocation in hypoxic myocytes. Blockade of Ca²⁺ entry and store refilling do not alter peak U46619 Ca²⁺ responses in either hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca²⁺ channels inhibits hypoxic augmentation of peak U46619 response. Ca²⁺ response to ryanodine alone is undetectable; ATP-induced Ca²⁺ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca²⁺ pool mobilization. Hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca²⁺, thromboxane hypersensitivity and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.