Neutral sphingomyelinases (N-SMases) are major candidates for stress-induced ceramide production, but there is still limited knowledge of the regulatory mechanisms of the cloned N-SMase enzyme-nSMase2. We have reported that p38 mitogen-activated protein kinase (MAPK) was upstream of nSMase2 in tumor necrosis-alpha (TNF-alpha)-stimulated A549 cells ( J Biol Chem 282: 1384-1396, 2007 ). Here, we report a role for protein kinase C (PKC) in mediating TNF-induced translocation of nSMase2 from the Golgi to the plasma membrane (PM). Pharmacological inhibition of PKCs prevented TNF-stimulated nSMase2 translocation to the PM in A549 cells. Using phorbol 12-myristate 13-acetate (PMA) as a tool to dissect PKC responses, we found that PMA induced nSMase2 translocation to the PM in a time- and dose-dependent manner. Pharmacological inhibitors and specific siRNA implicated the novel PKCs, specifically PKC-delta, in both TNF and PMA-stimulated nSMase2 translocation. However, PMA did not increase in vitro N-SMase activity and PKC-delta did not regulate TNF-induced N-SMase activity. Furthermore, PKC-delta and nSMase2 did not coimmunoprecipitate, suggesting that other signaling proteins may be involved. PMA-stimulated nSMase2 translocation was independent of p38 MAPK, and neither PKC inhibitors nor small interfering RNA had significant effects on TNF-stimulated p38 MAPK activation, indicating that PKC-delta does not act through p38 MAPK in regulating nSMase2. Finally, down-regulation of PKC-delta inhibited induction of vascular cell and intercellular adhesion molecules, previously identified as downstream of nSMase2 in A549 cells. Taken together, these data implicate PKC-delta as a regulator of nSMase2 and, for the first time, identify nSMase2 as a point of cross-talk between the PKC and sphingolipid pathways.