This study examined the role of ceramide generated by exogenous sphingomyelinases (SMases) on transcription nuclear factor-κB (NF-κB) activation and apoptosis in human colon epithelial HT-29 cells. Exogenous neutral (N) and acidic (A) SMase activated NF-κB with different kinetics, accounting for the diverse pattern of DNA binding of NF-κB complexes activated by tumor necrosis factor-α (TNF). NSMase activated predominantly RelA/p52 and RelA/p50 dimers within 30 min, while ASMase activated the p50/p50 homodimer by 20 h. The predominant activation of RelA-containing κB complexes by TNF or NSMase paralleled the induction of interleukin-8. HT-29 cells were sensitive to ASMase and TNF but resistant to NSMase. However, the apoptotic potential of NSMase was masked by NF-κB, as its prior inactivation sensitized HT-29 cells to NSMase. Thus, the generation of ceramide by exogenous SMases participates differentially in inflammation and apoptosis.