Background. 5-Fluorouracil (5-FU) causes intestinal mucosal damage and malabsorption. We have recently reported that coadministration of 17S,20-dimethyl-trans-2-prostaglandin E1 (OP-1206), a stable synthetic analogue of prostaglandin E1, with 5-FU to rats protects the small intestine from 5-FU-induced damage. Enterocyte proliferation would contribute to the restitution of the wounded intestinal mucosa. Thus, we investigated the effect of OP-1206 on the proliferation of rat jejunal crypt cells (IEC-6 cells) treated with 5-FU. Methods. Proliferation of IEC-6 cells was evaluated in terms of [3H]-thymidine incorporation and using the 3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Mucosal healing was assessed by measuring the speed of resealing across the denuded area of an IEC-6 cell monolayer. Results. OP-1206 stimulated [3H]-thymidine incorporation into subconfluent IEC-6 cells pretreated with 5-FU and increased the number of IEC-6 cells. AH23848B, an EP4 prostaglandin receptor antagonist, blocked the OP-1206-stimulated [3H]-thymidine incorporation into IEC-6 cells. The speed of resealing across the denuded area of a wounded IEC-6 cell monolayer was found to increase following treatment with OP-1206. Conclusions. OP-1206 stimulated the proliferation of IEC-6 cells treated with 5-FU, indicating a possible mechanism for the protective effect of OP-1206 against 5-FU-induced damage to the small intestine. OP-1206 was shown to be active in intestinal mucosal healing.