Objective To determine the optimal technique for establishing a chemically?induced oral ulcer model in rats for future treatment studies. Methods A total of 50 Sprague Dawley rats were randomly divided into five groups; groups 1, 2, 3, and 4 received 40%, 50%, or 60% acetic acid, or sodium hydroxide crystals, respectively, applied to the lower labial mucosa. All animals then received 50% acetic acid applied twice over the same area of the lower labial mucosa after the mucosa ulcer had healed. In the group 5, 50% acetic acid was applied to the buccal mucosa using a similar approach to examine the effects on formation of oral ulcers in different mucosal areas. The gross appearance of the mucosa, the ulcer formation rate, and the healing time were measured. Mucosal specimens were also taken to assess the histological features of the mucosal ulcer. Results Administration of 50% acetic acid to the lower labial mucosa produced the highest rate of ulcer formation, which showed a regular shape that allowed accurate measurement. The healing time and histological features of the ulcers in the groups 2 were similar to humans. By contrast, after the ulcers had healed, there was a low rate of new ulcers when applying 50% acetic acid over the lower labial gingival mucosa. Conclusions Oral ulcer induced by 50% acetic acid at the lower labial mucosa of Sprague Dawley rats is optimal for therapy evaluation. In addition, mucosa previously burnt by chemical regents are unsuitable for repeated establishment of an ulcer model.