Acarbose 1) (Figure 1) was isolated from the fermentation broth of Actinoplanes as a pseudooligosaccharide α-glucosidase inhibitor and serves as a clinically useful drug for the treatment of type II insulin-independent diabetes. The core unit of this compound responsible for the pharmacological function of α-glucosidase inhibition consists of an unsaturated cyclitol connected to a hexose via a nitrogen. The structure of the unsaturated cyclitol is identical with valienamine which is also found in other secondary metabolites, such as the amylostatins2), adiposins3), trestatins4), validamycins5) and salbostatin6) isolated from Streptomyces species. Biosynthetic studies have demonstrated that the cyclitol moieties of acarbose7) and validamycin8) are derived from the pentose phosphate pathway, presumably via sedoheptulose 7 phosphate or ido-heptulose 7-phosphate as intermediate.Many studies have been reported on the effects of nitrogen, carbon, phosphate, trace elements, or temperature on antibiotic production and cell growth in Actinomycetes9). In the case of acarbose, production occursonly in the presence of certain carbon sources such as maltose or higher oligosaccharides10)• Furthermore, it was found recently that there are two metabolic routes from maltotriose to the maltose unit of acarbose11). However, the source of the nitrogen of acarbose and the mechanisms of its introduction into a biosynthetic precursor of acarbose and of the linkage between the cyclitol and the deoxyhexose moieties have not yet been characterized. In this paper, therefore, we report the results of feeding experiments using various 15N-labeled compounds to determine the source of the nitrogen of acarbose, predicting the mechanism of the nitrogen introduction into a biosynthetic intermediate of acarbose.