Several prostaglandins are cytoprotective for the stomach; they prevent mucosal necrosis and hemorrhages produced by noxious agents, such as absolute ethanol. One possible mechanism of cytoprotection would be that the prostaglandin may prevent penetration of the necrotizing agent into the gastric mucosa. To test this hypothesis, 2 ml of 100% ethanol containing tracer amounts of 14C at carbon 1 was given orally to rats, after ligating the pylorus. [14C]Ethanol was measured in the gastric mucosa and in plasma from 2.5 to 60 min after ethanol administration. 16,16-Dimethyl prostaglandin E2 was given orally at a cytoprotective dose (10 micrograms/kg) 15 min before 100% ethanol. The level of [14C]ethanol (disintegrations per minute per gram of tissue) in the gastric mucosa of 16,16-dimethyl prostaglandin E2-treated animals were not different from those of control animals. The plasma levels were slightly lower during the first 10 min, but the area under the curve for the entire 60 min was the same in both groups. We conclude that (a) 16,16-dimethyl prostaglandin E2 does not prevent entry of ethanol into the gastric mucosa; (b) 16,16-dimethyl prostaglandin E2 protects the cells located deep in the gastric mucosa from necrosis, in spite of the fact that these cells are in contact with as much ethanol as cells of untreated animals; (c) gastric cytoprotection is probably due to a defense mechanism at the cellular level. These findings minimize the importance of luminal factors, such as an increase in mucus or bicarbonate, in the mechanism of cytoprotection.