Exogenous C(2)-ceramide has been shown to inhibit polymorphonuclear leukocyte (PMN) phagocytosis through inhibition of phospholipase D (PLD) and downstream events, including activation of extracellular signal-regulated kinases 1 and 2, leading to the hyphothesis that the sphingomyelinase pathway is involved in termination of phagocytosis. Here it is postulated that increased PLD activity generating phosphatidic acid and diacylglycerol (DAG) is essential for superoxide release and degranulation and that ceramide, previously shown to be generated during PMN activation, inhibits PLD activation, thereby leading to inhibition of PMN function. When PMNs were primed with granulocyte colony-stimulating factor (G-CSF) and then activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C(2)-ceramide (10 microM) completely inhibited release of superoxide, lactoferrin, and gelatinase; the DAG analog sn-1,2-didecanoylglycerol (DiC10) (10 microM) restored oxidase activation and degranulation in the ceramide-treated cells. Similarly, C(2)-ceramide inhibited oxidase activity and degranulation of PMNs treated with cytochalasin B followed by FMLP, and DiC10 restored function. In contrast, C(2)-ceramide did not inhibit phosphorylation of p47phox or p38 mitogen-activated protein kinase, or translocation of p47phox, PLD-containing organelles, adenosine diphosphate-ribosylation factor 1, RhoA, protein kinase C (PKC)-beta or PKC-alpha to the plasma membrane in G-CSF or cytochalasin B-treated, FMLP-activated PMNs. PLD activity increased by 3-fold in G-CSF-primed PMNs stimulated by FMLP and by 30-fold in cytochalasin B-treated PMNs stimulated by FMLP. Both PLD activities were completely inhibited by 10 microM C(2)-ceramide. In conclusion, superoxide, gelatinase, and lactoferrin release require activation of the PLD pathway in primed PMNs and cytochalasin B-treated PMNs. Ceramide may affect protein interactions with PLD in the plasma membrane, thereby attenuating PMN activation.