The dimeric enzyme sucrase-isomaltase (a complex of sucrose alpha-glucohydrolase, EC 3.2.1.48 and oligo-1,6-glucosidase (dextrin 6 alpha-D-glucanohydrolase), EC 3.2.1.10) of the rat small intestinal microvillus membrane is synthesized as a single chain enzymatically active precursor protein. This precursor (called pro-sucrase-isomaltase) was purified from fetal intestinal transplants in which sucrase-isomaltase was found almost exclusively in the uncleaved precursor form. A two-step procedure was developed using monoclonal antibody affinity chromatography on protein A Sepharose CL-4B followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequence of purified pro-sucrase-isomaltase was identical with that of the isolated isomaltase subunit which possesses the membrane anchor for the mature enzyme complex but differed from the NH2-terminal sequence of the sucrase subunit. This identity shows that the isomaltase domain comprising the membrane anchor is synthesized prior to the bulk of the protein destined to be localized on the luminal side of the microvillus membrane. A model is proposed for the mode of membrane assembly and the subsequent cleavage of pro-sucrase-isomaltase into its mature subunits.