BACKGROUND & AIMS: The short-chain fatty acid butyrate induces cell cycle arrest, differentiation, and apoptosis in colon cancer cells, but often induces opposite effects in normal colonic epithelial cells. We determined whether response to butyrate is dependent on the basal differentiation status of colonic epithelial cells. METHODS: Caco-2 cells at progressive stages of differentiation were treated with butyrate, and endpoints were measured. RESULTS: Response of Caco-2 cells to butyrate was dependent on their differentiation status. Butyrate maximally stimulated cell cycle arrest, apoptosis, alkaline phosphatase activity, transepithelial resistance, cell migration, urokinase receptor expression, and interleukin 8 secretion in undifferentiated Caco-2 cells, whereas differentiated Caco-2 cells were essentially resistant to these effects. Consistently, butyrate selectively induced histone hyperacetylation in undifferentiated Caco-2 cells. This resistance was also observed during HT29cl.19A cell differentiation, but not in the nondifferentiating SW620 cell line. Finally, the rate of butyrate use significantly increased as Caco-2 cells underwent spontaneous differentiation. CONCLUSIONS: Colonic epithelial cells become progressively more refractory to the effects of butyrate during absorptive cell differentiation. We postulate that this resistance is caused by the rapid use of butyrate by differentiated Caco-2 cells, which likely results in low intracellular concentrations and subsequently in its inability to inhibit histone deacetylase.