Tumor-promoting phorbol esters have been shown to modulate a number of physiological events in various cell types, and these events are associated with the activation of protein kinase C. The purpose of this study was to examine the effects of phorbol ester stimulation on protein kinase C activity and epithelial ion transport in the Cl−secreting rabbit corneal epithelium. We report here that nanomolar concentrations of tumor-promoting phorbol ester, 12-0-tetradecanoyl-phorbol-13 acetate (TPA), stimulate active ion transport via a putative receptor located in the corneal epithelium. This stimulation is a Cl−dependent process, but is independent of 6-adrenergic receptor activation and prostaglandin formation. Biochemical data support the idea that TPA activates Ca++/phospholipid-dependent protein kinase C in the corneal epithelium. Therefore, we suggest that protein kinase C may have a role in the regulation of membrane C1−transport in the mammalian corneal epithelium.