The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the enterocytes, we have conducted a computer-assisted cis-element search of the proximal human ALPI promoter sequence. A putative recognition site for the transcription factor hepatocyte nuclear factor (HNF)-4 was predicted at the positions from -94 to -82 in relation to the translational start site. The ability of HNF-4alpha to stimulate the expression from the ALPI promoter was investigated in the nonintestinal Hela cell line. Cotransfection with an HNF-4alpha expression vector demonstrated a direct activation of the ALPI promoter through this -94 to -82 element. EMSA showed that HNF-4alpha from nuclear extracts of differentiated intestinal epithelial cells (Caco-2) bound with high affinity to the predicted HNF-4 binding site. A 521 bp promoter fragment containing the HNF-4 binding site demonstrated a differentiation-dependent increase in promoter activity in Caco-2 cells. The presence of the HNF-4 binding site was necessary for this increase to occur.