The mechanism of chloride uptake at the basal membrane (stromal side) of rabbit corneal epithelium was examined by observing the effects of ion transport inhibitors and ion concentrations on the stimulated epithelial short-circuit current (Isc). Loop diuretics inhibited the theophylline-stimulated peak and sustained Isc. Treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.2 mM) and/or 5-(N,N-dimethyl)amiloride (0.1 mM) as well as the potent anion exchange inhibitor, 5c(+)[(2,3,9,9a-tetrahydro-1H-fluoren-7-yl)oxy]acetic acid (0.01 mM), had no significant effect on Isc. These results are consistent with Cl- uptake by a Na+-Cl- or Na+-K+-2Cl- cotransport mechanism rather than Cl(-)-HCO3(-)-OH- exchange coupled to Na+-H+ exchange. Incubation in low [Na+] or [Cl-] before stimulation with forskolin (0.1 mM) reduced both peak and sustained Isc, and saturation kinetics were exhibited. Hill coefficients for [Na+] and [Cl-] were 0.99 and 1.04, respectively, for peak Isc and 0.66 and 1.18, respectively, for sustained Isc. Apparent ion affinities for Na+ and Cl- were 13.5 and 18 mM, respectively, for peak Isc and 15 and 22 mM, respectively, for sustained Isc. These results favor Cl- uptake by a 1 Na+:1 Cl- cotransport mechanism for the rabbit corneal epithelium, but involvement of K+ in this process has not been eliminated.