Different digestive enzymes and transporters are present in the duodenum, jejunum, and ileum, but the factors determining region-specific gene expression are not yet understood. Homeobox transcription factors are important in defining gradients of cellular differentiation. The aim of this study was to investigate whether their expression differs between proximal (duodenal) and distal (ileal) regions of human small intestine. Intestinal RNA was prepared from surgical patients, and reverse-transcription polymerase chain reactions (PCRs) performed with mixed sequence oligonucleotide primers based on conserved regions. PCR products were identified by cloning and sequencing. Transcript abundance was determined by Northern blotting. The human homologues were identified as Cdx-1, Cdx-2 (or Cdx-3), Pdx-1 (previously named Islet/duodenal homeobox [Idx]-1, Ipf-1, or Stf-1), and 13 human homeodomain cluster genes, including HOXB3, HOXB4, and HOXA6. The relative abundance of some of these differed between duodenum and ileum. Pdx-1 transcripts were found only in duodenum, Cdx-2, Cdx-1, and HOXB3 were readily detectable in both regions, with Cdx-1 having a more marked distal expression. Many homeobox genes are expressed in human adult small intestinal mucosa, and some are found predominantly in one region.