Plasma membrane (PM) vesicles of defined sidedness were obtained from Beta vulgaris L. and subjected to limited proteolysis to investigate the topology and subunit composition of UDP-glucose: (1,3)-beta-glucan (callose) synthase (CalS). Latency experiments demonstrated that protease-sensitive sites on the CalS complex are located primarily at the cytoplasmic face of the PM, with little or no CalS inactivation occurring as the result of proteolysis at the apoplastic face. In the PM-bound form, CalS activity was resistant to inactivation by Pronase E, however at least four polypeptides previously implicated as possible CalS components (92, 83, 57 and 43 kDa) were extensively hydrolyzed. Polypeptides of 31, 29 and 27 kDa resisted Pronase E hydrolysis and were also enriched in CalS fractions purified by glycerol gradient centrifugation and product entrapment. In contrast to PM-bound CalS, purified CalS was rapidly hydrolyzed by Pronase E, indicating that most Pronase E-sensitive sites are deeply embedded within the PM. This study provides direct biochemical evidence that hydrophobic integral membrane proteins oriented primarily towards the cytoplasmic face of the PM are important for callose biosynthesis in Beta. Furthermore, these results form the basis of a biochemically derived working model largely consistent with morphologically derived models proposed for intramembrane PM-bound, microfibril-synthesizing complexes in higher plants.