We reported recently that ventromedial hypothalamic (VMH) lesions increased the synthesis of DNA in the gastrointestinal tract of rats by the firing of vagus nerve activity, mainly via cholinergic receptor mechanisms. In the present study, we examined whether the mitotic response is due to proliferation of a cell population--mucosal, submucosal, or muscular layer. A monoclonal antibody to proliferating cell nuclear antigen (PCNA) has previously been shown to be capable of identifying proliferating cells. Samples of formalin-fixed gastrointestinal epithelium, taken before and after VMH lesioning, were immunostained with the anti-PCNA monoclonal antibody, and the labeling index (LI) was determined. To discriminate the effect of hyperphagia in VMH lesioned rats, we utilized the method of pair-feeding. Cell proliferation was examined by the PCNA-labeling technique 0, 1, 3, and 7 days after VMH lesioning. The increase in proliferation was confined to cells in the mucosa and did not involve the muscularis and serosa. Studies in control animals showed that the LI was higher in the small intestine than in other gut segments, and higher in the large intestine than in the stomach. The mean PCNA-LI began to increase at 1 day and continued to increase for 3 days, then decreased 7 days following the lesioning. Results indicate that the gastrointestinal mucosa is in a state of hyperproliferation after VMH lesioning.