Plant mitochondrial NAD-malic enzyme (NAD-ME), which is composed of alpha- and beta-subunits in many species, participates in many plant biosynthetic pathways and in plant respiratory metabolism. However, little is known about the properties of woody plant NAD-MEs. In this study, we analyzed four NAD-ME genes (PtNAD-ME1 through PtNAD-ME4) in the genome of Populus trichocarpa. PtNAD-ME1 and -2 encode putative alpha-subunits, while PtNAD-ME3 and -4 encode putative beta-subunits. The Populus NAD-MEs were expressed in Escherichia coli cells as GST-tagged fusion proteins. Each recombinant GST-PtNAD-ME protein was purified to near homogeneity by glutathione-Sepharose 4B affinity chromatography. Milligram quantities of each native protein were obtained from 1 L bacterial cultures after cleavage of the GST tag. Analysis of the enzymatic properties of these proteins in vitro indicated that alpha-NAD-MEs are more active than beta-NAD-MEs and that alpha- and beta-NAD-MEs presented different kinetic properties (V-max, k(cat) and k(cat)/K-m). The effect of different amounts of metabolites on the activities of Populus alpha- and beta-NAD-MEs was assessed in vitro. While none of the metabolites evaluated in our assays activated Populus NAD-ME, oxalacetate and citrate inhibited all alpha- and beta-NAD-MEs and glucose-6-P and fructose inhibited only the alpha-NAD-MEs.