The activation of sorghum NADP-malate dehydrogenase is initiated by thiol/disulfide interchanges with reduced thioredoxin followed by the release of the C-terminal autoinhibitory extension and a structural modification shaping the active site into a high efficiency and high affinity for oxaloacetate conformation. In the present study, the role of the active site arginines in the activation and catalysis was investigated by site-directed mutagenesis and arginyl-specific chemical derivatization using butanedione. Sequence and mass spectrometry analysis were used to identify the chemically modified groups. Taken together, our data reveal the involvement of Arg-134 and Arg-204 in oxaloacetate coordination, suggest an indirect role for Arg-140 in substrate binding and catalysis, and clearly confirm that Arg-87 is implicated in cofactor binding. In contrast with NAD-malate dehydrogenase, no lactate dehydrogenase activity could be promoted by the R134Q mutation. The decreased susceptibility of the activation of the R204K mutant to NADP and its increased sensitivity to the histidine-specific reagent diethylpyrocarbonate indicated that Arg-204 is involved in the locking of the active site. These results are discussed in relation with the recently published NADP-MDH three-dimensional structures and the previously established three-dimensional structures of NAD-malate dehydrogenase and lactate dehydrogenase.