Microvillous atrophy is a disorder within the intractable diarrhea of infancy syndrome. The disease is believed to stem from a transport defect that prevents exocytosis of brush border-related material. We investigated this hypothesis by examining the direct constitutive exocytotic pathway using sucrase-isomaltase as a representative protein. We also studied various other brush border and lysosomal marker enzymes. The biosynthesis and localization of selected intestinal epithelial enzymes were studied in small-intestinal mucosal biopsy specimens from a total of nine children with microvillous atrophy by: (a) metabolic labeling in organ culture, (b) radioiodination and immunoprecipitation, (c) indirect immunoperoxidase immunocytochemistry, and (d) immunogold electron microscopy. The results demonstrated that brush border enzymes were synthesized normally and could be located in the apical brush border membrane and on microvillous membrane within microvillous inclusions. Brush border enzymes were not detected in the "secretory granules" that accumulated within the apical cytoplasm of epithelial cells. Lysosomal enzymes were only detected within lysosomal bodies. Thus, the direct constitutive pathway is not involved in microvillous atrophy, and a disturbance of endocytosis or the indirect constitutive pathway is unlikely. Any transport defect in the disease probably involves a different, unidentified exocytotic pathway.