The effect of several potentially harmful agents in gastric juice and duodenal contents on esophageal mucosa was investigated in the presence or absence of luminal H+ using an in vitro technique. Isolated rabbit esophageal mucosae were incubated in an Ussing chamber, and during exposure to the test agents mucosal integrity was assessed by measurement of mucosal potential difference (PD), tissue electrical resistance (R), and when luminal acid was present, the tissue permeability to H+. At pH 3.5 taurocholate caused a marked decrease in the PD and R and a substantial increase in the rate of luminal H+ loss. This effect was dependent on the presence of luminal H+, since no changes were observed with taurocholate at pH 7.4. In contrast, two of the three deconjugated bile salts tested at pH 7.4, deoxycholate and chenodeoxycholate, caused a profound reduction in the PD and R, similar to that observed with taurocholate in the presence of H+. Pepsin in the presence of H+, lysolecithin to a lesser degree, and trypsin in the absence of H+ also adversely affected isolated esophageal mucosa, but the effects were less pronounced than those caused by bile salts. Our data suggest that different mechanisms are operative in the pathogenesis of acidic and alkaline reflux esophagitis. In the presence of gastric H+, pepsin, and conjugated bile salts are the substances responsible for the greatest injury. In contrast, when acid is absent, trypsin and especially deconjugated bile salts are more crucial pathogenetic factors.