The purpose of the present study was to understand the mechanisms of action of diquafosol, a stable derivative of uridine 5′-triphosphate, on Cl– transport across the isolated rabbit conjunctiva. Rabbit conjunctivas were isolated and mounted in a modified Ussing chamber. Under short-circuit conditions, the effects were determined of mucosal (tear) side diquafosol application on the short-circuit current (Isc). Diquafosol rapidly and dose-dependently increased the Isc at concentrations ranging from 0.1 to 968 µM when added to the mucosal side of the conjunctiva. In the absence of the serosal Cl–, the Isc induced by 10 µM diquafosol was substantially reduced. On the contrary, in the absence of mucosal side Na+, the diquafosol-induced increases in Isc were unchanged. Following 45-min preincubation, the P2Y2 antagonist suramin inhibited the diquafosol-induced increases in the Isc whereas the P2Y1 antagonist pyridoxal-phosphate-6-azophenyl-2′4′-disulfonic acid had no effect. These studies suggest that diquafosol stimulates net Cl– secretion from the serosal to the mucosal side via stimulation of P2Y2 receptors in the rabbit conjunctiva.