12(R)-HETE [12(R)-hydroxy-5,8,10,14 eico-satetraenoic acid] is one of the major arachidonic acid metabolites produced by microsomal cytochrome P450 of the corneal epithelium. This metabolite is a potent inhibitor of Na+-K+-ATPase activity in several tissues. We investigated endogenous production of 12(R)-HETE in the rabbit corneal epithelium. Incubation of corneal epithelial sheets (prelabeled with14C-arachidonic acid) with arginine vasopressin resulted in the production of radioactive 12(R)-HETE suggesting its formation from endogenously labeled arachidonic acid. The maximal response was obtained with 1μM arginine vasopressin and represents a 15-fold increase in 12(R)-HETE formation compared with that of control tissues. Stimulation of14-arachidonic acid release with a detergent, digitonin, also resulted in endogenous 12(R)-HETE formation. Analysis of the incubation media following digitonin treatment of prelabeled corneal epithelial sheets revealed that 12(R)-HETE production was maximal at 20μM digitonin, a 17-fold increase over control values. This study is the first to describe hormonal and traumatic stimulation of 12(R)-HETE formation from endogenously labeled arachidonic acid in intact corneal tissues. This study demonstrates that the formation of this Na+-K+-ATPase inhibitor can be modulated by physiological and pathophysiological regulation.