Bile salts disrupt a functional gastric mucosal barrier which normally minimizes back-diffusion of H+ into mucosa. Our previous studies have shown that ionized bile salts disrupt the barrier to H+ by dissolving membrane lipids. The presence of an unstirred water layer on the surface of the gastric mucosa could protect against bile salt injury either by creating a concentration gradient of bile salt from lumen to mucosal surface or by slowing diffusion of lipid-laden mixed micelles away from the mucosal surface. In the present study we investigated this possibility in the anesthetized rat. Measurements of H+ back-diffusion and Na+ forward-diffusion across the gastric mucosa were made before and after exposure to a bile salt solution that was either unmixed or mixed by continuous withdrawal and injection. Using carbon monoxide diffusion, we observed this method of mixing to decrease the unstirred layer thickness from 880 to 448 μm (p < 0.02). Mixing increased mean H+ back-diffusion induced by a 10 mM mixture of six conjugated bile salts from -2.58 to -4.11 μEq/min (p < 0.01) and increased mean forward-diffusion of Na+ from 1.81 to 3.27 μEq/min (p < 0.01). Mixing also increased efflux of mucosal phospholipid (32.7 to 52.2 nmol/min, p < 0.05) and of cholesterol (4.89 to 8.87 nmol/min, p < 0.05) into the bile salt solution. Addition of saturation amounts of lecithin and cholesterol to the bile salt solution completely prevented disruption of the barrier whether the solution was mixed or not. Mixing also increased mucosal uptake of bile salt from 74.6 to 221.3 nmol/min (p < 0.01) when no lipids were added. In the presence of lecithin and cholesterol, mixing increased absorption of bile salt from 63.5 to 165.6 (p < 0.02). These findings further support the hypothesis that bile salts disrupt the gastric mucosal barrier by dissolution of mucosal membrane lipids, and provide evidence that the unstirred water layer helps protect the gastric mucosa from bile salt injury.