Rabbit corneal endothelial cells mounted in vitro were impaled simultaneously with Na(+)-selective and conventional KCl-filled microelectrodes. The membrane potential (Vm) was -30.4 +/- 0.8 mV (mean +/- SEM, n = 55) and the intracellular [Na+]i (calculated from the Na(+)-selective electrode potential, VNa) was 13.7 +/- 1.9 mM (mean +/- SEM, n = 16). When ouabain was added to the perfusate the cell depolarised, causing both Vm and VNa to increase with a very similar time course. Final Vm was -6.3 +/- 0.6 mV (mean +/- SEM, n = 15), and the final [Na+]i was 114 +/- 6.9 mM (mean +/- SEM, n = 5). The parallel increase in Vm and rise in [Na+]i suggest that a component of the ouabain-induced depolarisation of the cell (increase in Vm) is due to Na+ entry into the cell down its concentration gradient. The lateral and basal location of the Na+/K(+)-ATPase in bovine endothelial cells was confirmed (for the first time at the electron-microscopic level) using a monoclonal antibody specific for the alpha 1 subunit of Na+/K(+)-ATPase. The absence of a net Na+ flux across these cells combined with the basolateral location of the ATPase suggest that Na+ exit from the cell, and its re-entry take place across the same membrane (i. e. the basolateral).