2,5-Dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) is a pyrrolidine alkaloid that was isolated from the plant, Lonchocarpus sericeus. In the present study, DMDP was tested as an inhibitor of glycoprotein processing. MDCK cells were infected with influenza virus and the virus was raised in the presence of various amounts of DMDP. The glycoproteins were labeled by the addition of [2-3H]mannose or [1-3H]galactose to the medium. The virus was isolated by differential centrifugation and treated with Pronase to obtain glycopeptides. These glycopeptides were isolated by chromatography on Bio-Gel P-4, then digested with endoglucosaminidase H (Endo H) and rechromatographed on the Bio-Gel P-4 column. In the control virus, more than 70% of the glycopeptides were resistant to Endo H and were previously characterized as complex types of oligosaccharides. The remaining 20-25% are sensitive to Endo H and are of the high-mannose type. However, in the presence of DMDP (250 micrograms/ml), more than 80% of the glycopeptides are susceptible to digestion by Endo H. The oligosaccharide released by this treatment sized like a hexose11-12GlcNAc on a calibrated column of Bio-Gel P-4, and was only slightly susceptible to alpha-mannosidase treatment. This oligosaccharide was also labeled in the glucose moieties by growing the virus in [1-3H]galactose in the presence of DMDP. Following isolation, the oligosaccharide was subjected to complete methylation. Acid hydrolysis of the methylated oligosaccharide gave three methylated glucose derivatives, corresponding to 2,3,4,6-tetramethylglucose, 3,4,6-trimethylglucose, and 2,4,6-trimethylglucose in almost equal amounts. These data indicate that the oligosaccharide is a Glc3Man8-9-GlcNAc and that DMDP inhibits glucosidase I. Similar results were obtained with the cellular glycoproteins. DMDP did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it inhibit virus production as measured by plaque counts or hemagglutination assays. DMDP did cause some inhibition of mannose incorporation into the lipid-linked monosaccharides, but incorporation into lipid-linked oligosaccharides was not greatly affected, and incorporation into protein was stimulated. These results suggest that the pyrrolidine alkaloids are a new class of processing inhibitors.