Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays important roles in cell migration, cell proliferation and cell survival. Because these processes participate in the restoration of tubular integrity in renal ischaemia and reperfusion, FAK expression and phosphorylation at Tyr-397, the latter indicative of its activity, were examined in the different kidney zones by Western blot analysis and immunohistochemistry. Expression and phosphorylation of FAK were also studied in Madin-Darby canine kidney (MDCK) and medullary thick ascending limb (mTAL) cells after ATP depletion and repletion. In control rat kidneys, FAK expression in outer and inner medulla exceeded that in cortex, and phosphorylation of FAK at Tyr-397 was most pronounced in the inner medulla. Although this expression pattern was not affected by 20 (40, 60)-min ischaemia and 20 (40, 60)-min ischaemia followed by 60-min or 24-h reperfusion, FAK phosphorylation was significantly reduced in all kidney zones immediately after ischaemia, but increased during reperfusion, exceeding control values in the outer and inner medulla. ATP depletion and repletion of MDCK and mTAL cells were associated with a decrease in FAK phosphorylation during ATP depletion, followed by an increase during repletion. Rephosphorylation of FAK after ATP repletion was enhanced by N-acetylcysteine, a reactive oxygen species scavenger. ATP depletion disrupted focal adhesions in MDCK cells. Their reformation after ATP repletion paralleled the increase in FAK phosphorylation. These findings suggest an essential role for FAK-signalling during renal ischaemia and early reperfusion.