The mucus layer continuously covering the gastric mucosa consists of a loosely adherent layer that can be easily removed by suction, leaving a firmly adherent mucus layer attached to the epithelium. These two layers exhibit different gastro-protective roles; therefore, individual regulation of thickness, as well as mucin composition, was studied. Mucus thickness was measured in vivo with micropipettes in anesthetized mice (isoflurane, C57Bl/6, Muc1-/-, iNOS-/-, nNOS-/-) and rats (inactin) after surgical exposure of the gastric mucosa. The two mucus layers covering the gastric mucosa were differently regulated. Luminal administration of PGE2 increased the thickness of both layers, whereas luminal NO stimulated only firmly adherent mucus accumulation. A new gastro-protective role for iNOS was indicated, as iNOS deficient mice had thinner firmly adherent mucus layers and a lower mucus accumulation rate, and nNOS did not appear to be involved in mucus secretion. Down-regulation of gastric mucus accumulation was observed in Muc1-/- mice. Both the firmly and loosely adherent mucus layers consisted of Muc5ac mucins. In conclusion, this study showed that even though both the two mucus layers covering the gastric mucosa consist of Muc5ac, they are differently regulated by luminal PGE2 and NO. A new gastro-protective role for iNOS was indicated as iNOS-/- mice had thinner firmly adherent mucus layer. In addition, a signalling role of Muc1 was demonstrated, as down-regulation of gastric mucus accumulation was observed in Muc1-/- mice.