The basolateral Na-K-2Cl cotransporter (NKCC1) is a key component of the intestinal crypt cell secretory apparatus. Its fate during the transition to absorptive enterocyte and the potential impact of its altered expression on secretory output have not been addressed. In this report, NKCC1 mRNA was found to be expressed in rat jejunal crypt but not villus cells. Butyrate treatment of intestinal epithelial HT29 cells induced a differentiation pattern that recapitulated the rat intestinal crypt-villus axis, with NKCC1 mRNA levels decreasing in a time- and dose-dependent fashion in parallel with upregulation of apical brush-border markers. Butyrate but not acetate or proprionate decreased basal and cAMP-stimulated bumetanide-sensitive K+ (86Rb) uptake in both HT29 cells and the Cl--secreting T84 line. Butyrate markedly decreased transepithelial Cl- secretion in confluent T84 monolayers without effect on cAMP-regulated apical Cl- efflux. We conclude that NKCC1 regulation during enterocyte differentiation occurs at the level of gene expression, and that selective downregulation of NKCC1 gene expression and function by butyrate leads to a profound decrease in transepithelial Cl- secretion. These data emphasize the importance of NKCC1 in determining epithelial secretory capacity and suggest the possibility of modulation of the enterocytic transport phenotype as therapy for diarrheal disorders.