Ceramide is an important lipid second messenger produced by sphingolipid metabolism in cells exposed to a limited number of agonists and in turn triggers several cell responses in a protein kinase C (PKC)-dependent manner. Stimulation of mesangial cells with a radioiodinated photoaffinity labeling analogue of ceramide, (N-[3-[[[2-(125I)iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benz yl] oxy]carbonyl]propanoyl]-D-erythro-sphingosine) ([125I]-TID-ceramide), defines PKC-alpha and PKC-delta as direct _targets of ceramide. No binding of ceramide to PKC-epsilon and PKC-zeta could be detected. Moreover, TID-ceramide selectively binds to recombinant PKC-alpha and -delta but not to PKC-epsilon and -zeta isoenzymes. In vitro kinase activity assays reveal that only the binding of ceramide to PKC-alpha is accompanied by an increase in kinase activity. In contrast, there is no change in in vitro kinase activity of the other isoforms tested, i.e., PKC-delta, -epsilon, and -zeta, toward any of the conventional substrates tested. However, it is noteworthy that PKC-delta shows a decreased autophosphorylation upon ceramide binding. In vivo, activation of PKC-alpha by ceramide is monitored by a delayed translocation of the isoform from the cytosol to the membrane fraction, detectable after 1 h of stimulation. In contrast, neither PKC-delta, nor -epsilon nor -zeta is redistributed by ceramide. One functional cell response mediated by PKC-alpha in mesangial cells is a negative feedback regulation of ligand-stimulated phosphoinositide hydrolysis. When cells are pretreated with ceramide, ATP-induced inositol trisphosphate formation is time-dependently reduced. A maximal inhibition is observed after 2 h of ceramide exposure. In summary, these results suggest that ceramide selectively interacts with the alpha- and delta-isoforms of PKC in mesangial cells. Whereas PKC-alpha is activated with pronounced inhibition of hormone-stimulated phosphoinositide signaling, PKC-delta displays a decrease in its autophosphorylation, suggesting a negative role of ceramide binding on PKC-delta activity.