Sodium removal from the solution bathing the isolated rabbit corneal endothelium caused a reduction in both unidirectional and net flux of bicarbonate, whereas chloride withdrawal from the solution had no effect on the net bicarbonate flux but increased the unidirectional fluxes. These data correlate with previously published data on the effects of similar solution manipulations on both fluid movement and potential difference across the endothelium and strongly implicate bicarbonate as the primary ion involved in the maintenance of corneal hydration. Carbonic anhydrase (1 mg/ml) added to the solution bathing both sides of the endothelium markedly increased unidirectional and net bicarbonate fluxes, possibly by maintaining a high bicarbonate/CO2 concentration close to the membrane and thereby eliminating chemical gradients in the unstirred layer adjacent to the membrane. Determinations of intracellular pH with the 5,5-dimethyl-2,4-oxazolidine-dione method indicate that at more acid ambient conditions there is a lesser gradient between cell and bathing medium for H+; similar ambient conditions in other experiments resulted in larger unidirectional bicarbonate fluxes than at neutral pH. The data are suggestive of a nonvectorial H+-HCO3- exchange occurring across the endothelial cellular membranes.