Functional peptides have been produced by proteolysis of glucoamylase (glucan 1,4-α-glucosidase; EC 18.104.22.168) from Aspergillus niger and purified by affinity chromatography, gel filtration and two ion-exchange-chromatography steps. The peptides correspond to residues 499-616 and 509-616 of the original glucoamylase molecule. Together with GIC (residues 471-616 from glucoamylase 1) [Belshaw and Williamson (1990) FEBS Lett. 269, 350-353], the three peptides all contain the C-terminal domain (residues 509-616) but, in addition, contain different proportions of the O-glycosylated region. The properties of these peptides have been compared to define the function of the O-linked oligosaccharides in this protein. The O-glycosylated region plays only a minor role in binding to hydrogen-bond ordered starch. The difference between the apparent free energy (ΔG) for binding between the non-glycosylated C-terminal domain (-26.0 kJ/mol) and the C-terminal domain containing the fully O-glycosylated region (-25.0 kJ/mol) is only 1.0 kJ/mol. Binding to β-cyclodextrin suggests that even this difference may reflect a small conformational change in the C-terminal domain rather than a direct effect of the O-linked sugars. The c.d. spectrum of the O-glycosylated region is deduced by comparison of the three peptides and is predominantly that of a random-coil structure. Two-dimensional n.m.r. spectra of glucoamylase and of the glycosylated peptide 499-616 show that the binding domain is more mobile than the catalytic domain and that its mobility is further increased on removal of the catalytic domain. The O-glycosylated region is more mobile still, and there is a marked increase in its mobility on removal of the catalytic domain. The O-glycosylated region in the intact protein can therefore be envisaged as a semi-rigid rod. The results show that a major function of O-glycosylation in glucoamylase 1 is to provide an extended peptide backbone and hence a fixed distance in linking the catalytic and binding domains. It does not in itself significantly increase the binding affinity for starch.