An immortalized dorsal root ganglion cell line F‐11 exhibits many properties of spinal cord neurons and undergoes apoptosis in response to growth factor withdrawal and the exogenous addition of inhibitors of phosphatidylinositol‐3‐kinase (PI3K). To elucidate the mechanism of apoptosis we generated F‐11 clones which overexpressed either the p110 subunit of PI3K, a constitutively active form of protein kinase B/Akt (Myristoylated Akt), or a dominant‐negative form (c‐Akt). The first two constructs were protective against apoptosis induced by PI3K inhibitors such as wortmannin and LY294002. Caspase‐3 (CPP32) levels peaked at 4 hr to 6 hr in response to pro‐apoptotic drugs, and this increase was attenuated by 50% in F‐11 with constitutively active Akt. The Akt protection was confirmed by DNA fragmentation studies. Both neo‐transfected and the c‐Akt dominant‐negative transfected F‐11 cells showed increased ceramide formation (twofold) in response to staurosporine, wortmannin, or LY294002; whereas cells with a constitutively active Akt (Myr‐Akt) showed no increase in ceramide when treated with staurosporine, wortmannin, or LY294002. Ceramide was a more potent activator of CPP32 and an inducer of apoptosis when added as the native form (hydroxy‐ or nonhydroxy‐), rather than the more water‐soluble C₂‐ceramide. Overexpression of PI3K (p110) and Akt protected cells against ceramide‐induced apoptosis, suggesting that Ceramide action is upstream of Akt in these cells and suggesting that Akt might be a target for inhibition by ceramide. Both staurosporine and C₂‐ceramide activated the Jun kinase (JNK) cascade and C₂‐ceramide increased caspase‐3 (CPP32) activity in cells expressing wild‐type c‐Jun, but not dominant‐negative (TAM‐67) c‐Jun. We suggest that this pathway is also involved in apoptosis, consistent with the idea that ceramide has multiple kinase and kinase‐modulating targets in the apoptotic pathway of neurons. J. Neurosci. Sci. 57:884–893, 1999. © 1999 Wiley‐Liss, Inc.