Type I IFNs, like IFN-alpha, are major immune response regulators produced and released by activated macrophages, dendritic cells, and virus-infected cells. Due to their immunomodulatory functions and their ability to induce cell death in tumors and virus-infected cells, they are used therapeutically against cancers, viral infections, and autoimmune diseases. However, little is known about the adverse effects of type I IFNs on nondiseased tissue. This study examined the effects of IFN-alpha on cell death pathways in renal proximal tubular cells. IFN-alpha induced apoptosis in LLC-PK1 cells, characterized by the activation of caspase-3, -8, and -9, DNA fragmentation, and nuclear condensation. IFN-alpha also caused mitochondrial depolarization. Effector caspase activation was dependent on caspase-8 and -9. In addition to apoptosis, IFN-alpha exposure also decreased renal epithelial barrier function, which preceded apoptotic cell death. Caspase inhibition did not influence permeability regulation while significantly attenuating and delaying cell death. These results indicate that IFN-alpha causes programmed cell death in nondiseased renal epithelial cells. IFN-alpha-induced apoptosis is directed by an extrinsic death receptor signaling pathway, amplified by an intrinsic mitochondrial pathway. Caspase-dependent and -independent apoptotic mechanisms are involved. These findings reveal a novel aspect of IFN-alpha actions with implications for normal renal function in immune reactions and during IFN-alpha therapy.