The biosynthesis in vivo of rat intestinal sucrase—isomaltase [a complex of sucrose α-glucohydrolase, EC 3.2.1.48, and oligo-1,6-glucosidase (dextrin 6-α-D-glucanohydrolase), EC 3.2.1.10] has been studied by following the incorporation of L-[6-³H]fucose into the enzyme with time. Immunoprecipitation of sucrase—isomaltase from Triton-X-100-solubilized Golgi or basolateral membranes and subsequent polyacrylamide gel electrophoresis revealed the presence of an immunoreactive glycoprotein with an apparent molecular weight approximately twice that of the separated sucrase—isomaltase subunits, but no active subunits were found in these membranes. This glycoprotein was also found in the microvillus membrane in addition to the subunits of sucrase—isomaltase. Kinetic studies showed a maximal labeling of this glycoprotein in Golgi membranes at 15 min, in basolateral membranes at 30 min, and in microvillus membranes at 45 min and a half-life of less than 30 min in each membrane. However, the radioactivity of the sucrase—isomaltase subunits in the microvillus membrane reached a plateau after 60 min. These data suggest that sucrase—isomaltase is synthesized as a one-chain polypeptide precursor that is split into the subunits after its transfer to the microvillus membrane. Elastase (EC 3.4.21.11), but not trypsin (EC 3.4.21.4) or α-chymotrypsin (EC 3.4.21.1), split the putative precursor into two polypeptides that had electrophoretic behaviors similar to those of the active enzyme subunits. These studies suggest that pancreatic proteases may play an important role in the late posttranslational processing of sucrase—isomaltase in vivo.