1. A technique has been developed for measuring thickness of the gastric surface mucus gel layer. Mucosal sections (1·6 mm) were cut from frog and rat stomach and human antrum, mounted transversely and viewed by an inverse microscope (× 200 magnification) under dark field illumination or phase contrast. The mucus layer was readily distinguishable and its dimensions could be recorded by means of an eyepiece graticule. 2. Mean mucus gel thickness in rat, frog ( Rana temporaria and Rana pipiens ) and human was 73, 76, 55 and 192 μm respectively. However, there was variation in the average thickness of the gel layer between individual mucosae from the same species (up to twofold). Mucus thickness between adjacent regions of the same mucosal section also varied markedly (up to tenfold). 3. Topical administration of 16,16-dimethylprostaglandin E 2 by oral intubation caused a significant increase in thickness in both rat and frog at doses of 5 μg/ml and 0·5 μg/ml respectively. Feeding and exposure of the mucosa to N -acetylcysteine (10–20%, w/v) produced variable effects, whereas pepsin (1 mg/ml) caused a marked reduction in thickness of the surface gel layer in both rat and frog. 4. The technique provides a rapid and simple method for determining gastrointestinal mucus thickness in relation to mucosal morphology. It is ideally suited for studying the control of mucus secretion and effect of drugs.