After ischemia and reperfusion, severe alterations in the cytoskeletal organization of renal tubular epithelial cells have been reported. These effects, accompanied by a modification in the polarized distribution of some membrane transport proteins, are especially evident in the proximal tubule. In normal proximal tubule cells, actin is concentrated in apical brush border microvilli, along with the actin-binding protein villin. Because villin plays an important role in actin bundling and in microvillar assembly but can also act as an actin-fragmenting protein at higher calcium concentrations, we examined the effects of ischemic injury and reperfusion on the distribution of villin and actin in proximal tubule cells of rat kidney. Using specific antibodies against villin and actin, we show that these proteins redistribute in parallel from the apical to the basolateral plasma membrane within 1 h of reperfusion after ischemia. Ischemia alone had no effect on the staining pattern. Repolarization of villin to the apical membrane begins within hours after reperfusion with enhanced apical localization over time during the period of regeneration. This apical repolarization of villin is accompanied by the migration of actin back to the apical membrane. These results show not only that villin may be involved in the initial disruption of the actin cytoskeleton during reperfusion injury but also that its migration back to the apical domain of these cells accompanies the reestablishment of a normal actin distribution in the brush border.