Exposure of the rabbit corneal surface to a 20-microM digitonin-0.9% NaCl solution leads to permeabilization of the most superficial cells of the stratified epithelium. The devitalized cells exfoliate spontaneously from the corneal surface. Detergent exposure limited to 4-8 min leads to permeabilization and rapid exfoliation of a monolayer of surface cells. Consistent with the presence of the epithelial paracellular permeability barrier in this cell layer, their permeabilization results in complete loss of transepithelial resistance (Rt). Within minutes after detergent removal an initial recovery of Rt can be noticed indicating generation of a new paracellular permeability barrier by the viable sub-surface cells. This recovery proceeds rapidly and Rt reaches within 70 min a maximum equal to greater than 90% of the preexfoliation values (= 2.43 k omega.cm2, n = 22). The Rt recovery is fully blocked in a reversible manner by 10 microM dihydrocytochalasin B. The recovery is not affected by inhibition of protein synthesis with 5 microM cycloheximide. When the ocular surface is treated again with digitonin the permeabilization and exfoliation of a monolayer of cells and loss of Rt are repeated. After the second detergent exposure an initial recovery of Rt occurs as before within minutes. However, the pace of Rt recovery is much slower: 4-5 hr are required to reach a stable maximal Rt values amounting to about 73% of initial control. This recovery can be fully blocked by 5 microM cycloheximide indicating that protein synthesis is required for generation of tight junctions by the second subcellular layer. With only a fraction of Rt recovered, short-circuit currents amounting to, at least, 50% of control values and attributable in part to cell-to-tear movement of Cl- through the apical surface can be measured. This suggests that apical-type Cl- channels are either present in the apically facing membrane of subsurface cells or that they are rapidly inserted in it from preexisting intracellular pools immediately following the devitalization of the surface cells by digitonin.