Human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) cleaved synthetic vasoactive intestinal peptide (VIP1-28) with time-and peptidase concentration-dependence, which left less than 30% intact after 30 micrograms was incubated at 37 degrees C with 0.1 micrograms and 10 micrograms of peptidase for 120 min and 15 min, respectively. The rank order of relative rates of peptidolysis amino-terminal to hydrophobic amino acids was Ala4 and Val5 greater than Tyr22 and Ile26 much greater than Leu13 and Met17. The many effects of VIP1-28 on epithelial cell and leukocyte functions thus may be influenced by degradation of the mediator by enkephalinase at the surface of target cells.