Sodium butyrate is a well-recognized differentiating agent inducing alkaline phosphatase activity, one of the epithelial differentiation markers. When IEC6 cells, a nontransformed, small intestinal epithelial cell line, were cultured with butyrate, this substrate induced alkaline phosphate activity in a time- and dose-dependent fashion. However, the type of isoenzyme involved was a liver-type, not an intestinal-type. Electron microscopy revealed that the induced activity was strictly localized in the cytosol and not on the plasma membrane. However, disaccharidase activities, another kind of differentiation marker, were also enhanced by sodium butyrate. In addition, the positive cells demonstrating the presence of alkaline phosphatase activity were preferentially observed in tubular structures. These data show that butyrate-induced alkaline phosphatase activity is closely associated with differentiation-like phenomena in IEC6 cells, although the type of isoenzyme and cellular localization of the activity are different from those observed in mucosa.