Acute renal failure can occur after an ischemic injury and results in significant mortality. The stress-signaling pathways that are activated during renal ischemia are unknown. PP2A has emerged as an important regulator of cell death. To study the role of PP2A in ischemia-induced cell death, we used an in vitro model of simulated ischemia. In the present study, simulated ischemia in rat renal tubule epithelial NRK-52E cells (A) results in cell death that involves both necrosis and apoptosis, (B) activates PP2A, and (C) up-regulates the PP2A B56 alpha regulatory subunit. Previous data have shown that PKC alpha negatively regulates B56 alpha protein expression. Consistent with this finding, simulated ischemia suppressed PKC alpha and up-regulated B56 alpha. Treatment of NRK-52E cells with ceramide suppressed PKC alpha and activated PP2A in a manner that mimicked simulated ischemia. A role for PP2A in simulated ischemia-induced cell death is likely since inhibition of PP2A protected NRK-52E cells. In addition, overexpression of exogenous B56 alpha but not B55 in NRK-52E cells enhanced simulated ischemia-induced cell death. These findings suggest that activation of a PP2A isoform that contains the B56 alpha regulatory subunit is required for ischemia-induced cell death in kidney epithelial proximal tubule cells.