Starch debranching enzymes (DBE) are required for mobilization of carbohydrate reserves and for the normal structural organization of storage glucan polymers. Two isoforms, the pullulanase-type DBEs and the isoamylase-type DBEs, are both highly conserved in plants. To address DBE functions in starch assembly and breakdown, this study characterized the biochemical activity of ZPU1, a pullulanase-type DBE that is the product of the maize Zpu1 gene. Assays showed directly that recombinant ZPU1 (ZPU1r) expressed in Escherichia coli functions as a pullulanase-type enzyme, and 1H-NMR spectroscopy demonstrated that ZPU1r specifically hydrolyzes alpha(1-->6) branch linkages. Preferred substrates for ZPU1r hydrolytic activity were determined, as were pH, temperature, and thermal stability optima. Kinetic properties of ZPU1r with respect to two substrates, beta-limit dextrin and pullulan, were determined. ZPU1 activity was increased by incubation with thioredoxin h, and native activity was decreased in mutants that accumulate soluble sugars, suggesting potential regulatory mechanisms.