Mucus thickness is suggested to be related to mucosal protection. We therefore investigated the importance of the removable mucous layer and epithelial bicarbonate transport in preservation of the gastric juxtamucosal pH (pH_jm) during luminal acid. Anesthetized rats were prepared for intravital microscopy of the gastric mucosa, and pH_jm was measured with pH-sensitive microelectrodes. The mucus was either left intact (IM) or removed (MR) down to the firmly attached mucous layer, and HCl (pH 1) was applied luminally. Removal of the loosely adherent mucous layer did not influence the pH_jm during luminal acid (pentagastrin: IM/MR 7.03 ± 0.09/6.82 ± 0.19; pentagastrin + indomethacin: IM/MR 6.89 ± 0.20/6.95 ± 0.27; ranitidine: IM/MR 2.38 ± 0.64/2.97 ± 0.62), unless prostaglandin synthesis and acid secretion were inhibited (ranitidine + indomethacin: IM/MR 2.03 ± 0.37/1.66 ± 0.18). Neutral pH_jm is maintained during endogenous acid secretion and luminal pH 1, unless DIDS was applied luminally, which resulted in a substantially decreased pH_jm (1.37 ± 0.21). Neutral pH_jm is maintained by a DIDS-sensitive bicarbonate transport over the surface epithelium. The loosely adherent mucous layer only contributes to maintaining pH_jm during luminal pH 1 if acid secretion and prostaglandin synthesis are inhibited.