Activation, proliferation, or programmed cell death of T lymphocytes is regulated by the mitochondrial transmembrane potential (Δψ m) through controlling ATP synthesis, production of reactive oxygen intermediates (ROI), and release of cell death-inducing factors. Elevation of Δψ m or mitochondrial hyperpolarization is an early and reversible event associated with both T cell activation and apoptosis. In the present study, T cell activation signals leading to mitochondrial hyperpolarization were investigated. CD3/CD28 costimulation of human PBL elevated cytoplasmic and mitochondrial Ca 2+ levels, ROI production, and NO production, and elicited mitochondrial hyperpolarization. Although T cell activation-induced Ca 2+ release, ROI levels, and NO production were diminished by inositol 1, 4, 5-triphosphate receptor antagonist 2-aminoethoxydiphenyl borane, superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, spin trap 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide, and NO chelator carboxy-2-phenyl-4, 4, 5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide, mitochondrial hyperpolarization was selectively inhibited by carboxy-2-phenyl-4, 4, 5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide (− 85.0±10.0%; p= 0.008) and, to a lesser extent, by 2-aminoethoxydiphenyl borane. Moreover, NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate diethylenetriamine elicited NO and ROI production, Ca 2+ release, transient ATP depletion, and robust mitochondrial hyperpolarization (3.5±0.8-fold; p= 0.002). Western blot analysis revealed expression of Ca-dependent endothelial NO synthase and neuronal NO synthase isoforms and absence of Ca-independent inducible NO synthase in PBL. CD3/CD28 costimulation or H 2 O 2 elicited severalfold elevations of endothelial NO synthase and neuronal NO synthase expression, as compared with β-actin. H 2 O 2 also led to moderate mitochondrial hyperpolarization; however, Ca 2+ influx by ionomycin or Ca 2+ release from intracellular stores by thapsigargin alone failed to induce NO synthase expression, NO production, or Δψ m elevation. The results suggest that T cell activation-induced mitochondrial hyperpolarization is mediated by ROI-and Ca 2+-dependent NO production.