Recent studies have suggested that heat shock proteins (HSPs) are involved in the restoration of the cytoskeletal anchorage of Na, K-ATPase after renal ischemia. To determine their role in ischemic conditioning, we investigated whether cytoskeletal Na, K-ATPase was stabilized during repeat ischemia concurrent with 25-kD and 70-kD HSPs induction. Anesthetized rats either underwent single unilateral renal ischemia or were conditioned with bilateral renal ischemia and, after 18 h of reflow, were then subjected to repeat unilateral renal ischemia. Renal cortex was harvested, and effects of single versus repeat ischemia were compared by Triton X-100 extraction, by immunohistochemistry, and by an in vitro assay of Na, K-ATPase association with isolated cytoskeletal fractions. In contrast to single ischemia, repeat ischemia did not result in increased Triton X-100 extractability of Na, K-ATPase. Levels of 25-kD and 70-kD HSPs were significantly induced by ischemic conditioning and redistributed into the cytoskeletal fraction after single and repeat ischemia. Immunohistochemistry also showed significant disruption of Na, K-ATPase within proximal tubules only after a single episode of ischemia, whereas repeat ischemia did not alter the pattern of restored Na, K-ATPase localization in conditioned renal cortex. The preserved association of Na, K-ATPase with the cytoskeletal fraction of conditioned renal cortex was effectively abolished in vitro by addition of antibodies against 25-kD or 70-kD HSP. These results suggest that 25-kD and 70-kD HSPs induced by ischemic conditioning stabilize the cytoskeletal anchorage of Na, K-ATPase during repeat renal ischemia.