The objective of this study was to determine whether Na(+)-glucose cotransport contributed to the residual short-circuit current (Isc) in the pigmented rabbit conjunctiva not accounted for by Cl- secretion. The Isc of the pigmented rabbit conjunctiva was measured following exposure of its mucosal or serosal side to varying concentrations of D-glucose or phloridzin in a glutathione-bicarbonated Ringer's solution (GBR). Addition of D-glucose to the mucosal side of the conjunctiva bathed in glucose-free GBR elevated Isc up to 26 +/- 6% in a dose-dependent manner (K0.5 = 1.2 mM), while mucosal (but not serosal) addition of phloridzin reduced the Isc (IC50 = 0.05 mM) of the conjunctiva bathed in regular GBR. In a mucosal Na(+)-free medium, neither 20 mM D-glucose nor 0.5 mM phloridzin was effective. In a mucosal glucose-containing medium, deletion of Na+ reduced Isc up to 27 +/- 4%. It was, however, restored to its initial value upon the addition of 17.5-141 mM Na+ to a medium containing 5 mM D-glucose. Hill plot analysis of Isc elevation at 141 mM Na+ in the presence of varying D-glucose concentrations in the mucosal bathing fluid yielded a Hill coefficient of 0.86. There is, therefore, evidence for the apical localization of phloridzin-sensitive Na(+)-coupled D-glucose transport that exhibits apparent 1:1 stoichiometry, being responsible for the maximal 26% increase in the Isc.