The potential of the intestinal bile acid transporter to serve as a shuttle for small peptide molecules was investigated. Eleven peptides with a 2-6 amino acid backbone were conjugated to the 24-position of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oic acid (cholic acid) via an amide bond using an automated peptide synthesizer. In a human intestinal cell line (CaCo-2), cholic acid-peptide conjugates were able to inhibit the transepithelial transport of [3H]taurocholic acid, a natural substrate for the bile acid carrier, at a 100:1 conjugate/substrate ratio. Affinity for the carrier decreased significantly when the conjugate in the 24-position increased from 1 to 2 amino acids. Further increase in the amino acid chain length caused only minor decrease in affinity. A tetrapeptide-bile acid conjugate, [3H]-ChEAAA (Ch = cholic acid), was transported by the bile acid transporter, showing markedly higher apical (AP)-to-basolateral (BL) compared to BL-to-AP transport and inhibition by a 100-fold excess taurocholic acid. Another conjugate with 6 amino acids (ChEASASA) was transported by a passive diffusion pathway but still showed higher transport rates than the passive permeability marker mannitol, suggesting the possibility that the cholic acid moiety aids the passive membrane transfer of peptide molecules by increasing its lipophilicity. Metabolism of bile acid-peptide conjugates in CaCo-2 cells was 3% over 3 h. In conclusion, these studies show that the coupling of peptides to the 24-position of the sterol nucleus in cholic acid results in a combination of decreased metabolism and increased intestinal absorption, either by a carrier-mediated pathway or by accelerated passive diffusion.