Multiple studies have addressed the mechanisms by which ultraviolet (UV) light induces cell death, and a few have focused on stress mediators such as acid sphingomyelinase (ASMase) or protein kinase C (PKC). Based on a recent study that identified a novel mechanism of activation of ASMase through phosphorylation (1), the current study was undertaken to determine the upstream mechanisms regulating ASMase in response to UV and to investigate the role of ASMase and its phosphorylation at S508 as an integral event during UV light-induced cell death. Exposure of MCF-7 breast cancer cells to UV light type C (UVC) transiently activated ASMase with maximal activity detected at 10 min postirradiation. A significant increase in C₁₆-ceramide was detected concomitant with a decrease in C₁₆-sphingomyelin. In marked contrast, cells overexpressing the ASMase^S508A mutant, which could not be phosphorylated, had no change in either ASMase activity or ceramide levels post-UV radiation. Loss of PKC by RNA interference or its inhibition by rottlerin blocked ASMase phosphorylation and membrane targeting, thus implicating PKC upstream of ASMase activation by UV light. Further investigations revealed that UV radiation altered mitochondrial morphology from elongated tubules to fragmented perinuclear organelles, consistent with the onset of the apoptotic cascade. Importantly, cells overexpressing ASMase^S508A were protected (>50%) from UV light-induced mitochondrial fragmentation. Mechanistically, the results showed that ASMase^S508A cells had 50% less active Bax than ASMase^WT cells. These molecular differences culminated in resistance of ASMase^S508 cells to UVC-induced cell death (25%) as compared to ASMase^WT cells (46%). Taken together, this study provides key molecular insights into activation of ASMase in response to UV light, the role of PKC in this activation, and the role of ASMase in mediating apoptotic responses.—Zeidan, Y. H., Wu, B. X., Jenkins, R. W., Obeid, L. M., Hannun, Y. A. A novel role for protein kinase c-mediated phosphorylation of acid sphingomyelinase in UV light-induced mitochondrial injury.