Binding of eNOS to the chaperone protein, Hsp90, promotes coupled eNOS synthetic activity. Using resistance level pulmonary arteries (PRA) from 2-day, 5- to 7-day and 12-day old piglets, we tested the hypothesis that Hsp90/eNOS interactions are developmentally regulated in the early neonatal period. PRA were isolated for co-immunoprecipitation and immunoblot analyses or cannulated for continuous diameter measurements using the pressurized myography technique. NOS inhibition caused less constriction in PRA from 2-day compared with 5- to 7-day and 12-day old piglets. No age related differences were found in dilation responses to an NO donor or in protein expression of Hsp90, phospho-eNOS (Ser1177), AKT, phospho-AKT or caveolin-1. Compared to the older animals, PRA from 2-day old piglets had higher total eNOS expression but displayed less binding of eNOS to Hsp90 and AKT. Hsp90 antagonism with radicicol induced greatest constriction in PRA from 12-day old piglets. Acetylcholine (ACh) stimulation caused dilation in PRA from 5- to 7-day and 12-day old but not 2-day old animals, despite rapid and equivalent ACh-mediated eNOS phosphorylation (Ser1177) in all 3 age groups. Hsp90 inhibition abolished ACh-mediated dilation in PRA from the older piglets. ACh failed to stimulate Hsp90/eNOS binding in 2-day old but induced a significant increase in Hsp90/eNOS co-immunoprecipitation in PRA from the older age groups which was blocked by Hsp90 antagonism. We conclude that physical interactions between Hsp90 and eNOS mature over the first weeks of life, likely contributing to the postnatal fall in pulmonary vascular resistance and changes in agonist-induced pulmonary vascular responses characteristic of the early neonatal period.