Many metabolic reactions are coupled to NADPH in the mitochondrial matrix, including those involved in thiol group reduction. One enzyme linked to such processes is mitochondrial NADP+-dependent isocitrate dehydrogenase (mtICDH; EC 1.1.1.42), although the precise role of this enzyme is not yet known. Previous work has implicated mtICDH as part of a biochemical mechanism to reductively activate the alternative oxidase (AOX). We have partially purified mtICDH from tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) cell suspension cultures and localized this to a 46-kDa protein on SDS-PAGE, which was verified by peptide sequencing. In the inflorescence of the aroid Sauromatum guttatum Schott (voodoo lily), mtICDH appears to be developmentally regulated, presenting maximal specific activity during the thermogenic period of anthesis when the capacity for AOX respiration is also at its peak. Transgenic tobacco plants were generated that overexpress mtICDH and lines were obtained that demonstrated up to a 7-fold increase in mtICDH activity. In isolated mitochondria, this resulted in a measurable increase in the reductive activation of AOX in comparison with wild type. When examined in planta in response to citrate feeding, a strong conversion of AOX from its oxidized to its reduced form was observed in the transgenic line. These data support the hypothesis that mtICDH may be a regulatory switch involved in tricarboxylic acid cycle flux and the reductive modulation of AOX.