Mitochondrial functions have a major impact on T-cell functionality. In this study we characterized whether mitochondrial function in the neonatal T-cells differs from that in the adult T-cells during short T-cell activation. We used flow cytometry methods to test mitochondrial mass and to monitor mitochondrial Ca²⁺ levels, mitochondrial potential and superoxide generation in parallel with cytoplasmic Ca²⁺ levels during phythohaemagglutinine-induced activation of CD4+ and CD8+ T-cells of 12 term neonates and 11 healthy adults. Baseline mitochondrial mass of CD4+ and CD8+ cells was lower in the neonate than in the adult. In comparison with the adult, neonatal resting CD4+ T-cells had lower cytoplasmic Ca²⁺ levels and this was associated with normal activation induced Ca²⁺-response. During short-term activation cytoplasmic Ca²⁺-response was lower in neonatal than in adult CD8+ T-cells. Mitochondrial Ca²⁺ uptake was increased in CD4+ neonatal T cells while it decreased in CD8+ T-cells. Mitochondrial depolarization was increased in CD4+ and decreased in CD8+ neonatal T-cells compared to adults. Superoxide generation was higher and equal in neonatal CD4+ and CD8+ cells, respectively, compared to the adult ones. Our data suggest that neonatal T-cells exhibit marked differences in mitochondrial function and superoxide generation compared to adult T-cells.