Divergent results from in vitro studies on the thickness and appearance of the gastrointestinal mucus layer have previously been reported. With an in vivo model, we studied mucus gel thickness over time from stomach to colon. The gastrointestinal tissues of Inactin-anesthetized rats were mounted luminal side up for intravital microscopy. Mucus thickness was measured with a micropipette before and after mucus removal by suction. The mucus layer was translucent and continuous; it was thickest in the colon (∼830 μm) and thinnest in the jejunum (∼123 μm). On mucus removal, a continuous, firmly adherent mucus layer remained attached to the epithelial surface in the corpus (∼80 μm), antrum (∼154 μm), and colon (∼116 μm). In the small intestine, this layer was very thin (∼20 μm) or absent. After mucus removal, there was a continuous increase in mucus thickness with the highest rate in the colon and the lowest rate in the stomach. In conclusion, the adherent gastrointestinal mucus gel in vivo is continuous and can be divided into two layers: a loosely adherent layer removable by suction and a layer firmly attached to the mucosa.