Recently, Leunberger and Novikoff (1) have shown that the endothelial cells are the primary sites of Na+-K+-ATPase, a key mechanism in the control of hydration of cornea. Since this enzyme would be a useful marker for our study of endothelial damage by CO2 laser (2), we have undertaken a comparative study for the localization of this enzyme at ultrastructural level with different methods. In the course of this work we developed a new method with the use of 5-nitroindoxyl phosphate as a synthetic substrate. This new substrate, like p-nitrophenyl phosphate (3), meets the requirements for the demonstration of the K+ activated step of dephosphorylation of ATPase. In addition, it can be used with glutaraldehyde fixed tissue, thereby offering an important advantage over the previous method (3).Rabbit corneas were freshly excised under pentobarbitol anesthesia.