Nitration of both protein-bound and free tyrosine by reactive nitrogen species results in the formation of nitrotyrosine (NT). We previously reported that free NT impairs microtubule polymerization and uncouples endothelial nitric oxide synthase (eNOS) function in pulmonary artery endothelial cells (PAEC). Since microtubules modulate mitochondrial function, we hypothesized that increased NT levels during inflammation and oxidative stress will induce mitochondrial dysfunction in PAEC. PAEC isolated from fetal lambs were exposed to varying concentrations of NT. At low concentrations (1-10 µM), free NT increased nitration of mitochondrial electron transport chain (ETC) protein subunits complexes I-V and state III oxygen consumption. Higher concentrations of NT (50 µM) caused decreased microtubule acetylation, impaired eNOS interactions with mitochondria, and decreased ETC protein levels. We also observed increases in heat-shock-protein-90 (hsp90) nitration, mitochondrial superoxide formation, and fragmentation of mitochondria in PAEC. Our data suggest that free NT accumulation may impair microtubule polymerization and exacerbate reactive oxygen species-induced cell damage by causing mitochondrial dysfunction.