The production of 12-hydroxyeicosatetraenoic acid by the corneal epithelium of several species has been extensively reported yet the controversy over the exclusive production of the (S) epimer (a lipoxygenase-derived metabolite) endures. Incubation of calf corneal epithelial microsomes (3 mg/ml) with arachidonic acid and NADPH resulted in the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). The synthesis of 12-HETE was inhibited by SKF-525A and clotrimazole, selective inhibitors of cytochrome P450 dependent activities, but not by indomethacin or BW-755C, inhibitors of cyclooxygenase and lipoxygenase activities, respectively. Chiral analysis revealed the presence of both enantiomers; however, the R isomer was the predominant one, i.e., 91 +/- 5% vs. 9 +/- 5% for the R and S enantiomers, respectively. Since the R enantiomer is the product of a cytochrome P450-mediated reaction, it suggests that the major metabolic activity in these microsomes is cytochrome P450-dependent and supports the claim for cytochrome P450 reactions in this ocular tissue.