12(R)-Hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE], a cytochrome P450 arachidonate metabolite, is metabolized by corneal tissues via three distinct metabolic pathways: beta-oxidation, omega-hydroxylation, and keto-reduction. The major metabolite released from the intact rabbit corneal epithelium or cultured cells was identified by mass spectrometric analysis as 8-hydroxy-4,6,10-hexadecatrienoic acid, the tetranor metabolite derived following two steps of beta-oxidation from the carboxy terminus. The beta-oxidation pathway was expressed in both microsomes and mitochondria isolated from bovine corneal epithelium and was dependent on the addition of oxidizing equivalents. The major metabolite of 12(R)-HETE in subcellular fractions of bovine corneal epithelial cells was a dihydro compound, 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE). This derivative is presumably formed by an oxidation of the hydroxyl group followed by two keto-reduction steps, since its formation was accompanied by the appearance of a keto metabolite identified as 12-oxo-5,8,14-eicosatrienoic acid. The omega-hydroxylation, in contrast to other cell types, was a minor route for 12(R)-HETE metabolism in these tissues. Since 12(R)-HETE has been implicated as a modulator of Na(+)-K(+)-ATPase activity and its related functions in ocular tissues, these findings raise the possibility that the newly described metabolites may be involved in regulating corneal functions. In addition, the presence of a keto reductase in the cornea may be of great importance following injury since 12(R)-HETrE resulting from 12(R)-HETE by this activity is a potent ocular proinflammatory compound.