This study investigated the function of the starch synthase isoform SSIII in production of starch in maize kernels. Starch synthases (SS) catalyze reactions that build alpha-(1,4)-linked linear glucosyl chains, which are the constituents of the major starch polymer amylopectin (Ap). The five SS classes of plants are highly similar in the catalytic and starch-binding domains of the C-termini but differ at their N-termini, with SSIII having the longest N-terminal arm. In maize, SSIII is the product of dull1 (du1) gene. Most SSs affect Ap structure directly via their enzymatic activities, but much indirect evidence suggests that the SSIII isoform also regulates other starch metabolizing enzymes via protein-protein interactions. To investigate the function of SSIII in determination of starch structure, ten allelic du1' mutations were characterized with respect to the molecular lesion causing the mutation, changes in SS activity, structural changes to the Ap component of starch, and pleiotropic effects on other starch metabolizing enzymes. Although allelic differences in the extent of expression loss were observed, all ten du1 mutations had identical effects on Ap structure, producing Ap molecules with more short chains and fewer long chains. The notion that these structural changes are indirect effects of altered activities of DU1 binding partners is supported by evidence that isoamylase-type starch debranching enzyme activity is increased in all of the du1 mutant kernels. To examine potential regulatory effects of the SSIII N-terminus, transgenic maize plants were generated expressing full-length SSIII or either of two truncated versions containing specific regions of the amino terminal extension and lacking the catalytic domain. The glucan chain length distribution in amylopectin from the transgenic kernels expressing the complete N terminal extension of SSIII, disconnected from the catalytic domain, was distinct from that of either wild type or du1' mutants. Thus, the N terminal arm has been shown to regulate starch biosynthesis, independent of the catalytic activity of SSIII. The data are consistent with a model proposing that multiple enzymes of starch biosynthesis are coordinated through physical interactions involving the SSIII amino terminus.