Acute duodenal ulcer produced by subcutaneous injection of propionitrile in rats was studied by stereo, light, and electron microscopy in order to gain insight into the localization and mechanism of initial cell injury. Stereomicroscopy revealed an initial fissuring and splitting of the tips of the villus folds within 4 hours after two injections of propionitrile. This was followed by sloughing of the epithelium, shortening and effacement of the villus folds, and within 24 hours the appearance of discrete ulcers in the mucosa of the proximal duodenum. In most of the rats, two ulcers developed: the first and larger ulcer was on the antimesenteric side of the duodenum, and the other, a small and more superficial one, was on the opposite wall. Ultrastructural lesions appeared in the absorptive epithelial cells of the proximal duodenum within 5 hours following a single dose of propionitrile. The cytoplasmic changes of cellular injury were preceded and/or accompanied by beading and loss of microvilli. The initiation of propionitrile-induced alterations at the tip of the villi in the proximal duodenum suggests that the ulcerogenic agent acts from the luminal side of the duodenum and probably originates from the stomach.