Human neutrophils (PMN) stimulated by tumor necrosis factor (TNF) synthesize and release platelet-activating factor (PAF) transiently. In the present investigation, we have examined the mechanism responsible for the down-regulation of PAF synthesis. The response of PMN is proportional to the occupancy of high-affinity TNF receptors on the PMN plasma membrane, as shown by binding assays with [125I]TNF. These receptors are down-regulated within 10 min of the addition of TNF; the receptors reappear after 60 min, but the PMN do not resume PAF synthesis. Further PAF synthesis is obtained by adding acetyl-coenzyme A (CoA) to the culture medium. However, this compound does not diffuse into PMN, as shown by incubating these cells with labeled acetyl-CoA. This finding suggests that PAF synthesis is regulated by the amount of acetyl-CoA available to the lyso-PAF: acetyltransferase on the cell plasma membrane. This acetyl-CoA is accessible to chemicals in the culture medium, since it is hydrolysed by hydroxylamine. The inhibition of PAF synthesis by hydroxylamine is reversed by adding acetyl-CoA. PAF synthesis in TNF-treated cells appears to be regulated either by the amount of acetyl-CoA available or by the ability to transfer acetyl-CoA from the cellular pool to the lyso-PAF: acetyltransferase.