Limbal basal epithelium is thought to possess corneal epithelial stem cells that are the ultimate source of corneal epithelial proliferation and differentiation during corneal epithelial wound healing. Destruction of the limbal epithelium results in corneal conjunctivalization and vascularization, suggesting that the limbal epithelium also may be a barrier between corneal and conjunctival epithelia. In this experiment, a total corneal epithelial debridement using combined n-heptanol and mechanical scraping was created immediately (one-step) or 5 weeks (two-step) after 15 or 30 sec n-heptanol treatment at the limbus. All defects healed in 1-2 weeks. The severity of corneal vascularization, as judged by external photography, followed the ascending order of 30-sec two-step and 15-sec two-step less than 15-sec one-step less than 30-sec one-step (P less than 0.005). Immunofluorescence studies using monoclonal antibodies AM-3 and AE-5 showed mixed expression of corneal and conjunctival epithelial phenotypes on the corneal surface in the one-step subgroups. By contrast, the two-step subgroups had a normal corneal epithelial phenotype. Impression cytology was used to map goblet-cell distribution on the perilimbal corneal surface. The specimens taken from superior, temporal, and inferior bulbar areas were evaluated by a scoring system at different times. The extent of goblet cells invading onto the corneal surface also followed the same ascending order (P = 0.005). A transient goblet-cell surge was noted, and the extent was related to the extent of corneal vascularization. It is thus evident that in vivo n-heptanol treatment for different durations can result in different extents of corneal conjunctivalization and vascularization. The authors concluded that the capability of the remaining limbal basal epithelium to recover its original full-thickness stratified layers determines the strength of the limbal barrier.