The aim of this work was to evaluate the function of isoamylase in starch granule biosynthesis in Arabidopsis leaves. A reverse-genetic approach was used to knockout AtISA1, one of three genes in Arabidopsis encoding isoamylase-type debranching enzymes. The mutant (Atisa1-1) lacks functional AtISA1 transcript and the major isoamylase activity (detected by native gels) in crude extracts of leaves. The same activity is abolished by mutation at the DBE1 locus, which encodes a second isoamylase-type protein, AtISA2. This is consistent with the idea that ISA1 and ISA2 proteins are subunits of the same enzyme in vivo. Atisa1-1, Atisa2-1 (dbe1), and the Atisa1-1/Atisa2-1 double mutant all have identical phenotypes. Starch content is reduced compared with the wild type but substantial quantities of the soluble glucan phytoglycogen are produced. The amylopectin of the remaining starch and the phytoglycogen in the mutants are structurally related to each other and differ from wild-type amylopectin. Electron micrographs reveal that the phytoglycogen-accumulating phenotype is highly tissue-specific. Phytoglycogen accumulates primarily in the plastids of the palisade and spongy mesophyll cells. Remarkably, other cell types appear to accumulate only starch, which is normal in appearance but is altered in structure. As phytoglycogen accumulates during the day, its rate of accumulation decreases, its structure changes and intermediates of glucan breakdown accumulate, suggesting that degradation occurs simultaneously with synthesis. We conclude that the AtISA1/AtISA2 isoamylase influences glucan branching pattern, but that this may not be the primary determinant of partitioning between crystalline starch and soluble phytoglycogen.