Rationale: Cyclosporin A (CsA) is known to preserve cardiac contractile function during endotoxemia, but the mechanism is unclear. Increased nitric oxide (NO) production and altered mitochondrial function are implicated as mechanisms contributing to sepsis-induced cardiac dysfunction, and CsA has the capacity to reduce NO production and inhibit mitochondrial dysfunction relating to the mitochondrial permeability transition (MPT). Objectives: We hypothesized that CsA would protect against endotoxin-mediated cardiac contractile dysfunction by attenuating NO production and preserving mitochondrial function. Methods: Left ventricular (LV) function was measured continuously over 4 hours in cats assigned as follows: Controls (n=7); lipopolysaccharide (LPS) alone (3 mg/kg, n=8), and cyclosporin A (CsA, 6 mg/kg, n=7), a calcineurin inhibitor that blocks the MPT, or tacrolimus (FK506, 0.1 mg/kg, n=7), a calcineurin inhibitor lacking MPT activity, followed in 30 minutes by LPS. Myocardial tissue was then analyzed for NOS2 expression, tissue nitration, protein carbonylation and mitochondrial morphology and function. Measurements and Main Results: LPS treatment resulted in impaired LV contractility, altered mitochondrial morphology and function, and increased protein nitration. As hypothesized, CsA pre-treatment normalized cardiac performance and mitochondrial respiration and reduced myocardial protein nitration. Unexpectedly, FK506 pre-treatment had similar effects, normalizing both cardiac and mitochondrial parameters. However, CsA and FK506 pre-treatments markedly increased protein carbonylation in the myocardium despite elevated manganese superoxide dismutase activity during endotoxemia. Conclusions: Our data indicate that calcineurin is a critical regulator of mitochondrial respiration, tissue nitration, protein carbonylation and contractile function in the heart during acute endotoxemia.