Starch metabolizing enzyme isoforms are known to exhibit functional interactions. This research employed one and twodimensional starch zymograms to investigate pleiotropic enzymatic effects in starch branching enzyme (BE) and debranching enzyme (DBE) mutants of maize. Activity bands are shown to correspond with specific gene products by immunoblot analysis or biochemical assay. Multiple migratory forms are observed for BEI and BEIIa, and a single form for BEIIb. SU1 isoamylase-type DBE migrates as three distinct forms and ZPU1 pullulanase-type DBE as a single form. For each BE and DBE, a genetic null mutation conditions complete loss of all forms of the respective enzyme. The BE mutation amylose-extender also results in loss of two BEI migration forms, and the sul-starchy and zpul-204 DBE mutations result in a loss of BEIIa activity. In both DBE mutants, BEIIa is of normal abundance and size, indicating the effect is posttranslational and directly impacts enzyme function. Zymogram analysis of the uncharacterized opaque5 (o5) mutant shows o5 endosperm is deficient in β-amylase activity, providing insight into a biochemical defect resulting from the mutation. Finally, a novel enzyme activity identified in starch zymograms was characterized and found to be a glucan hydrolase of the β-amylase type. This research demonstrates that activity gel analysis is an effective approach for uncovering pleiotropic enzymatic effects that might not be discovered using RNA microarray or proteomics technologies, and indicates that some mutational effects on starch metabolizing enzymes are likely due to altered protein complex associations.