The Arabidopsis (Arabidopsis thaliana) genome contains four genes encoding putative NADP-malic enzymes (MEs; AtNADP-ME1-ME4). NADP-ME4 is localized to plastids, whereas the other three isoforms do not possess any predicted organellar targeting sequence and are therefore expected to be cytosolic. The plant NADP-MEs can be classified into four groups: groups I and II comprising cytosolic and plastidic isoforms from dicots, respectively; group III containing isoforms from monocots; and group IV composed of both monocots and dicots, including AtNADP-ME1. AtNADP-MEs contained all conserved motifs common to plant NADP-MEs and the recombinant isozymes showed different kinetic and structural properties. NADP-ME2 exhibits the highest specific activity, while NADP-ME3 and NADP-ME4 present the highest catalytic efficiency for NADP and malate, respectively. NADP-ME4 exists in equilibrium of active dimers and tetramers, while the cytosolic counterparts are present as hexamers or octamers. Characterization of T-DNA insertion mutant and promoter activity studies indicates that NADP-ME2 is responsible for the major part of NADP-ME activity in mature tissues of Arabidopsis. Whereas NADP-ME2 and -ME4 are constitutively expressed, the expression of NADP-ME1 and NADP-ME3 is restricted by both developmental and cell-specific signals. These isoforms may play specific roles at particular developmental stages of the plant rather than being involved in primary metabolism.