Epidermal growth factor (EGF) is produced in Brunner's glands and plays a role in healing and repair of duodenal ulcers. We examined the participation of zwitterionic phospholipids of mucus in the effects of EGF. Under anesthesia, groups of rats received an intraduodenal bolus of either saline or EGF. Some rats received subcutaneous indomethacin followed by EGF or EGF followed by a detergent (5% Brij 35, a nonionic detergent that solubilizes luminal phospholipids). Thirty minutes after treatment, mucosal surface hydrophobicity and phospholipid concentration in the mucus layer were measured. Matched groups of rats were challenged with 0.5 M HCl, instilled intraduodenally 30 min after treatment, and mucosal damage was assessed 1 h after acid challenge. Exogenous EGF significantly increased surface hydrophobicity and phosphatidylcholine concentration in the mucus layer. EGF treatment also reduced mucosal damage induced by acid. However, indomethacin pretreatment or detergent administration after EGF abolished both protection against acid and changes in the mucus layer. These data suggest that EGF increases duodenal resistance to luminal acid via stimulation of mucosal zwitterionic phospholipids.