Animal studies and clinical pediatric practice have shown that acute alkalosis attenuates hypoxic pulmonary vasoconstriction (HPV). However, increased intracellular pH appears to enhance pulmonary vasoreactivity. We therefore hypothesized that prolonged alkalosis augments HPV. This study compares the effects of acute and prolonged alkalosis on HPV in isolated perfused lungs of 1-month-old lambs (n = 5) and the hypoxic responses of 300- to 500-μm diameter segments of pulmonary arteries (n = 7) from mature cats at control pH and after 30 min of alkalosis. In isolated lamb lungs, normocarbic (5% CO2) hypoxia (4% O2) increased the total pressure gradient (ΔPT) by 6.0 ± 2.7 (SEM) mm Hg (p ≤ 0.05). Acute hypocarbia (3% CO2) increased the perfusate pH to ˜ 7.52 and significantly decreased the hypoxic ΔPT to normocarbic, normoxic (28% O2) levels. Subsequent exposure to normoxia (while maintaining alkalosis) further decreased ΔPT. However, re-exposure to hypoxia after 60 min of normoxic alkalosis significantly increased ΔPT by 11.6 ± 1.6 mm Hg (p ≤ 0.05) to a level similar to that seen during normocarbic hypoxia. The increased hypoxic reactivity (i.e., change in pressure between normoxia and hypoxia) during prolonged alkalosis was due to enhanced HPV of the small vessels within the middle segment of the pulmonary circuit, as defined by an inflow-outflow occlusion technique (p ≤ 0.05). The occlusion data also suggested that most of this increase occurred in small arteries. Moreover, the hypoxic response of isolated small arteries from the cat was increased almost threefold (p ≤ 0.05). While extrapolation to the clinical situation from in vitro animal studies must be done cautiously, the enhanced HPV seen after 30 to 60 min of normoxic alkalosis in these studies suggests the need for further evaluation of the consequences of prolonged alkalosis therapy in the management of pulmonary hypertension in neonates and children.