Enterocytes at the tips of microvilli are more sensitive to an ischemic insult than those cells residing in the crypts, an effect thought to be due to a relative lack of collateral flow. We speculated that this increased cellular sensitivity to ischemia might be an intrinsic feature of the cells related to their differentiated phenotype. To test this hypothesis, enterocyte response to ischemia was determined using both in vivo and in vitro models. For the in vivo studies, male Sprague-Dawley rats underwent laparotomy, and small intestinal ischemia was induced by clamping the superior mesenteric artery for 30 or 60 minutes, after which reperfusion was allowed for various time points up to 4 days. Injury was assessed histologically, as well as with Northern blots, probing for the enterocyte differentiation markers intestinal alkaline phosphatase and lactase, as well as the gut-epithelial marker villin. Mucosal changes consistent with ischemia/reperfusion injury were evident--that is, a rapid inflammatory response followed by progressive villus cell loss beginning at the tips and progressing to the crypts, depending on the degree of insult, with an eventual return to normal microanatomy. Intestinal alkaline phosphatase and lactase were lost immediately after ischemia and returned with reperfusion, confirming that the differentiated cells are particularly sensitive to ischemic injury. The in vitro studies employed two separate models of enterocyte differentiation: sodium butyrate-treated HT-29 cells and Caco-2 cells maintained for 7 days after confluence. In both models, undifferentiated and differentiated cells were subjected to treatment with 2-deoxyglucose and oligomycin-A (in vitro model of ischemia) and apoptosis was assessed by fluorescence-activated cell sorting analysis. Differentiation of both cell lines resulted in a significantly greater apoptotic response to ischemia compared to undifferentiated cells exposed to an identical insult. We conclude that differentiated enterocytes may be inherently more sensitive to ischemia-induced injury than their undifferentiated counterparts. These findings call into question the popularly held belief that villus tip cells are more susceptible to ischemia because of their location relative to the microvascular anatomy.