Mutant lines defective for each of the four starch debranching enzyme ( DBE) genes ( AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis ( Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase- type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water- soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch- excess phenotype at the end of both the light and the dark phases accompanied by a small modification! of the amylopectin structure. This result suggests that this isoamylase- type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single- mutant lines did not lead to a distinctive phenotype. However, Atisa2/ Atpu1 double- mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.