Platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3- phosphocholine) is a mediator involved in the pathogenesis of inflammatory diseases associated with tissue eosinophil infiltration. Previous studies utilizing bioassay or assaying enzymes associated with PAF biosynthesis have suggested that human eosinophils produce PAF. The present study has extended these initial studies by identifying and quantifying the different PAF molecular species and analogues synthesized by human eosinophils in response to A23187 and f-Met-Leu-Phe (FMLP). Gas chromatography-mass spectrometric analysis indicated that A23187-stimulated eosinophils produce at least three molecular species of PAF. The predominant species is 1-hexadecyl-2-acetyl-GPC (16:0) followed by 1-octadecyl-2-acetyl-GPC (18:0) and 1-octadecyl-2-acetyl-GPC (18:1). Eosinophils stimulated with FMLP produce approximately 100-fold smaller quantities of PAF relative to those produced in response to A23187 and only the 16:0 molecular species could be measured. A small percentage (comprising between 2 and 5%) of the 2-acetylated phospholipids produced by eosinophils was the 1-acyl analogue of PAF. Long-term (72 hr) incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in a three- to fourfold increase in PAF synthesis from eosinophils stimulated with FMLP, without changes in the profile of PAF molecular species or in the percentage of the 1-acyl analogue of PAF. These data indicate that human eosinophils can produce various molecular species of PAF and that this process can be quantitatively enhanced by GM-CSF.