The effect of castanospermine on the processing of N-linked oligosaccharides was examined in the parent mouse lymphoma cell line and in a mutant cell line that lacks glucosidase II. When the parent cell line was grown in the presence of castanospermine at 100 micrograms/ml, glucose-containing high-mannose oligosaccharides were obtained that were not found in the absence of inhibitor. These oligosaccharides bound tightly to concanavalin A-Sepharose and were eluted in the same position as oligosaccharides from the mutant cells grown in the absence or presence of the alkaloid. The castanospermine-induced oligosaccharides were characterized by gel filtration on Bio-Gel P-4, by h.p.l.c. analysis, by enzymic digestions and by methylation analysis of [3H]mannose-labelled and [3H]galactose-labelled oligosaccharides. The major oligosaccharide released by endoglucosaminidase H in either parent or mutant cells grown in castanospermine was a Glc3Man7GlcNAc, with smaller amounts of Glc3Man8GlcNAc and Glc3Man9GlcNAc. On the other hand, in the absence of castanospermine the mutant produces mostly Glc2Man7GlcNAc. In addition to the above oligosaccharides, castanospermine stimulated the formation of an endoglucosaminidase H-resistant oligosaccharide in both cell lines. This oligosaccharide was characterized as a Glc2Man5GlcNAc2 (i.e., Glc(1,2)Glc(1,3)Man(1,2)Man(1,2)Man(1,3)[Man(1,6)]Man-GlcNAc-GlcNAc). Castanospermine was tested directly on glucosidase I and glucosidase II in lymphoma cell extracts by using [Glc-3H]Glc3Man9GlcNAc and [Glc-3H]Glc2Man9GlcNAc as substrates. Castanospermine was a potent inhibitor of both activities, but glucosidase I appeared to be more sensitive to inhibition.