NADP-dependent malic enzymes (NADP-ME; EC1.1.1.40) have been implicated in a wide range of metabolic pathways in the plastids and cytosol of plant cells. In maize, an NADP-ME type C4 plant, the most abundant NADP-ME form is the chloroplastic leaf isoform that delivers CO2 intracellularly to ribulose bisphosphate carboxylase (RuBPCase). A second NADP-ME isoform predominates in maize roots and exhibits distinct C3-like enzymatic characteristics. We show that the C3-like isoform is encoded by a pair of nearly identical genes that encode precursor proteins with functional chloroplast transit peptides. Using RT-PCR, we also show that the messages encoding the C4 and C3-like NADP-ME isoforms are differentially regulated with respect to the developmental stage of the leaf, light conditions, and tissue type. Based on these characteristics and on sequence comparison of ME families in other species, we propose a scheme for the origin of the C4-specific NADP-ME gene.