The function of a putative galacturonosyltransferase from Arabidopsis (Arabidopsis thaliana; At1g02720; GALACTURONOSYLTRANSFERASE-LIKE5 [AtGATL5]) was studied using a combination of molecular genetic, chemical, and immunological approaches. AtGATL5 is expressed in all plant tissues, with highest expression levels in siliques 7 DPA. Furthermore, its expression is positively regulated by several transcription factors that are known to regulate seed coat mucilage production. AtGATL5 is localized in both endoplasmic reticulum and Golgi, in comparison with marker proteins resident to these subcellular compartments. A transfer DNA insertion in the AtGATL5 gene generates seed coat epidermal cell defects both in mucilage synthesis and cell adhesion. Transformation of atgatl5-1 mutants with the wild-type AtGATL5 gene results in the complementation of all morphological phenotypes. Compositional analyses of the mucilage isolated from the atgatl5-1 mutant demonstrated that galacturonic acid and rhamnose contents are decreased significantly in atgatl5-1 compared with wild-type mucilage. No changes in structure were observed between soluble mucilage isolated from wild-type and mutant seeds, except that the molecular weight of the mutant mucilage increased 63% compared with that of the wild type. These data provide evidence that AtGATL5 might function in the regulation of the final size of the mucilage rhamnogalacturonan I.