An increasing number of cell-surface receptors have been shown to trigger sphingomyelin turnover and generation of the lipid signaling molecule ceramide. Ceramide plays a role in mediating cellular responses as diverse as inflammation, differentiation, gene expression, growth suppression, and apoptosis. A radioiodinated, photoaffinity-labeling analog of ceramide ([125I]TID-ceramide) was used to identify downstream signaling targets of ceramide. Ceramide was found to bind specifically to and activate protein kinase c-Raf, leading to subsequent activation of the extracellular signal-regulated kinase (ERK) module in mesangial cells. We found also that ceramide binds to and differentially modulates the activity of distinct protein kinase C isoenzymes. These data are discussed in the context of interleukin 1beta-induced inflammatory gene expression in mesangial cells.