Abstract Studies in 1940 on potato phosphorylase reaction with starch found that d-glucopyranose from α-d-glucopyranosyl-1-phosphate was added to the nonreducing-ends of starch chains. This led to the hypothesis that the biosynthesis of starch required a preformed primer. Later it was found that phosphorylase was exclusively a degradative enzyme in vivo and that starch-synthase was the enzyme that reacted with ADPGlc to biosynthesize starch. Amylogenin, a putative self-glycosylated protein, was postulated to be the primer, although it was never demonstrated or found. In the present study, three reactions were performed in sequence with a highly purified potato starch-synthase to determine whether an amylogenin primer was present and required or whether the biosynthesis was de novo. Reaction 1 was performed by adding 2.0mM ADPGlc to synthesize the putative primer to a possible amylogenin in the preparation; in Reaction 2, 10mM ADP-[14C]Glc was added; and in Reaction 3, 10mM nonlabeled ADPGlc was added. After the isolation, reduction, and acid hydrolysis of the products of Reactions 2 and 3, 14C-d-glucitol was obtained from Reaction 2 and was decreased by Reaction 3. The formation of 14C-d-glucitol and its decrease showed that an amylogenin, protein primer was not involved in starch biosynthesis and the synthesis is de novo by the addition of d-glucose to the reducing-ends of growing starch chains.