Treatment of human neutrophils with tumor necrosis factor-alpha (TNF-alpha) resulted in an increase in concentration of ceramide and its catabolite, sphingosine. Sphingosine, a potent endogenous protein kinase C (PKC) inhibitor, as well as TNF-alpha, induced internucleosomal DNA fragmentation and morphological changes characteristic of apoptotic cells. Ceramide and sphingosine-1-phosphate, the initial product of sphingosine catabolism, did not cause apoptosis under our experimental conditions. In addition, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and N,N-dimethylsphingosine (DMS), known as PKC inhibitors, also induced apoptosis, suggesting that induction of apoptosis by sphingosine may be related to inhibition of PKC activity. These results indicate that sphingosine deacylated from ceramide may be an endogenous modulator mediating apoptotic signals by TNF-alpha in neutrophils.