The purpose of this study was to isolate the platelet-activating factor (PAF) precursor and other choline phosphoglycerides (GPC) i.e. the alkenylacyl and diacyl lipids from the rabbit cornea, to analyze their fatty acid content and to determine which pool was the most susceptible to arachidonate depletion when activated corneal tissue released arachidonic acid (AA) and metabolites. Rabbit iridal GPC was also analyzed for comparative purposes. The fatty acid methyl esters of the GPC components extracted from the rabbit cornea and iris-ciliary body, isolated by high performance liquid chromatography (HPLC), were determined by capillary gas liquid chromatography. Rabbit corneas were labelled in vivo by intracameral injection of3H-AA (1μCi, specific activity = 218 Ci / mmol) and cryogenically injured 18 h later. Corneas were incubated in vitro and the AA and eicosanoids released into the medium were extracted and separated by HPLC. The GPC was extracted from the tissues and the labeling of the three GPC constituents was quantified by liquid scintillation counting. The corneal and iridal PAF precursor represented 4.1±0.2% and 2.9±0.2% respectively of total GPC in those tissues. On a mole basis, the alkyl arachidonoyl species constituted 12.7±0.7% of the corneal and 38±0.6% of the iridal PAF precursors respectively. The release of AA and prostaglandins by the cornea was linear until 15 min; whereas 12-HETE levels continuously increased until 60 min. All GPC components lost label but 1-0-alkyl-2-arachidonoyl was the most affected, with its labeled content 50% less than the non-injured control. These results suggest that the PAF precursor could be an important source of bioactive lipids in the cornea after phospholipase A2activation induced by injury.