Experiments were conducted to determine if the Cl^- secretagogue, 1-ethyl-2-benzimidazolinone (EBIO), exhibits such activity in the rabbit conjunctival epithelium. For this, epithelia were isolated in an Ussing-type chamber under short-circuit conditions. The effects of EBIO on the short-circuit current (I_sc) and transepithelial resistance (R_t) were measured under control, physiological conditions, as well as, in experiments with altered electrolyte concentrations. Addition of 0.5 mM EBIO to the apical bath stimulated the control I_sc by 64% and reduced Rt by 21% (P< 0.05 as paired data). Under Cl^--free conditions, I_sc stimulations by EBIO were markedly attenuated. In the presence of an apical-to-basolateral K⁺ gradient and permeabilization of the apical membrane, the majority of the I_sc reflects the transcellular movement of K⁺ via basolateral K⁺ channels. Under these conditions, EBIO in combination with A23187 elicited nearly instantaneous 60-90% increases in I_sc that were sensitive to the calmodulin antagonist, calmidazolium, and the K⁺ channel blocker TEA. In the presence of an apical-to-basolateral Cl^- gradient and nystatin permeabilization of the basolateral aspect, EBIO increased the Cl^--dependent I_sc, an effect prevented by the channel blocker, glibenclamide (0.3 mM). The latter compound was also used to determine the proportion of EBIO-evoked unidirectional ³⁶Cl^- fluxes in the presence of the Cl^- gradient that traversed the epithelium transcellularly. Overall, EBIO activated apical Cl^- channels and basolateral K⁺ channels (presumably those that are Ca²⁺-dependent), thereby suggesting that this compound, or related derivatives, may be suitable as topical agents to stimulate fluid transport across the tissue in individuals with lacrimal gland deficiencies.