Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg2+ from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross-contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from com roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast-specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions.