We examined the inhibitory effect of three pseudo-aminosugars (validamine, valienamine, and valiolamine), which were isolated from the broth of Streptomyces hygroscopicus, on the oligosaccharide-processing glucosidases I and II involved in glycoprotein biosynthesis in rat liver. Both glucosidases I and II were inhibited to the same extent by the pseudoaminosugars, and valiolamine had a more potent inhibitory activity than validamine or valienamine. A 50% inhibition of valiolamine was observed at 12 /iM for glucosidase I and glucosidase II activities acting respectively on the substrates Glc3ManeGlcNAc2 and p-nitrophenyl a-D-glucopyranoside. Further, in order to investigate further the ability of valiolamine to inhibit glucosidase I, reaction products were analyzed by gel nitration on a Bio-Gel P-4 column. We also compared the inhibitory action of these pseudo-aminosugars on the acid a-glucosidase of rat liver lysosomes. They competitively inhibited the hydrolysis of both substrates, maltose and glycogen. Valiolamine again had a more potent lysosomal a-glucosidase inhibitory activity than the other two. The Kt values of valiolamine for the hydrolysis of maltose and glycogen were 8.1 and 11 /iM, respectively. Valiolamine is a particularly effective inhibitor of oligosaccharide glucosidases I and II and of lysosomal a-glucosidase. Hence valiolamine might be useful as a research tool in investigations of carbohydrate metabolism.