Although unesterified polyunsaturated fatty acids (PUFA) have been shown to elicit marked changes in neutrophil function, the associated signal transduction processes require clarification. In this study we examined the effect of PUFA on the sphingomyelin (SM)-signalling cycle in human neutrophils. Treatment of neutrophils with eicosatetraenoic acid [arachidonic acid, 20:4(n-6)] caused a decrease in the mass of cellular SM and an increase in the level of ceramide. 20:4(n-6)-stimulated neutral sphingomyelinase (SMase) activity of the leucocytes in a time- and concentration-dependent manner. Other unsaturated fatty acids, docosahexaenoic [22:6(n-3)], eicosapentaenoic [20:5(n-3)], octadecenoic [oleic, 18:1(n-9)] and octadecadienoic [linoleic, 18:2(n-6)] acids also had the capacity to activate neutral SMase; however, certain 20:4(n-6) derivatives ¿20:4(n-6) methyl ester [20:4(n-6)ME], 15-hydroperoxyeicosatetraenoic (15-HPETE) and 15-hydroxyeicosatetraenoic (15-HETE) acids¿, very-long-chain PUFA ¿tetracosatetraenoic [24:4(n-6)] and octacosatetraenoic [28:4(n-6)] acids¿ and saturated fatty acids [octadecanoic (stearic, 18:0) and eicosanoic (arachidic, 20:0) acids] had no significant effect. Activation of neutral SMase by 20:4(n-6) appeared to involve metabolism via 20:4(n-6)CoA (arachidonoyl CoA) and was not dependent on prostaglandin and leukotriene synthesis. All of the fatty acids and derivatives tested failed to activate acidic SMase of neutrophils. Ceramide was found to inhibit 20:4(n-6)-induced superoxide generation by the cells. It is envisaged that the PUFA-induced ceramide production in neutrophils plays a role in the regulation of biological responses.