When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, four polar metabolites (compounds A-D) were formed. Synthesis of these metabolites could be inhibited by carbon monoxide, SKF 525A, and anti-cytochrome c reductase antibodies. One of the metabolites, compound C, was found to inhibit partially purified Na+,K+-ATPase from the corneal epithelium in a dose-dependent manner with an ID5o of =50 nM. After compound C was purified by TLC and HPLC, it was found to have a UV absorption spectrum with a maximum absorbance at 236 nm suggesting the presence of a conjugated diene. Mass spectrometric analysis using positive-and negative-ionization modes was carried out on derivatized compound C that had been synthesized from a mixture of specifically labeled ([5,6,8,9,11,12,14, and unlabeled arachidonic acid. Abundant fragment ions were consistent with compound C being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon-12 of the icosanoid backbone; all deuterium atoms from [2Hgjarachi-donate were retained in the structure.