To examine directly the synthesis and intracellular processing of rat intestinal sucrase-isomaltase, [3H]leucine-labeled enzyme was solubilized from purified microsomal and microvillus fractions, immunoprecipitated, and analyzed. Immunologic identity between the microsomal and microvillus forms of the enzyme was demonstrated. Time-course studies using both intravenous and intraluminal labeling demonstrated an initial peak of microsomal sucrase-isomaltase labeling at 30 min followed by a sharp decline, and a subsequent rise in microvillus sucrase-isomaltase labeling, suggesting a precursor-product relationship. Fluorography and quantitative analysis of electrophoresed immunoprecipitates confirmed these results, and revealed the presence of two high molecular weight proteins in the microsomes. Susceptibility of the smaller high molecular weight precursor to cleavage with endo-beta-N-acetyl-glucosaminidase H indicated that this was the initially synthesized form of the glycoprotein. These data are consistent with the membrane flow hypothesis and the single polypeptide model suggested for sucrase-isomaltase synthesis.