Neurons can be damaged by the activation of glutamate receptors, but whether glutamate is related to the development of glaucomatous optic neuropathy is still controversial. The purpose of this study was to measure the acute changes in the glutamate levels in the optic nerve head (ONH) of rabbits induced by an artificial elevation of the intraocular pressure (IOP). A concentric microdialysis probe was inserted into the ONH of rabbits via the pars plana. The probe was perfused with Ringer's solution, and the levels of glutamate in 10-min dialysate samples were measured repeatedly using high-performance liquid chromatography. After the glutamate level was stabilized for at least 60 min, the IOP was adjusted to three levels; 120 mm Hg (n=11), 60 mm Hg (n=12), and 15 mm Hg (control group; n=11). The IOP was altered by changing the height of a bottle of Ringer's solution, which was connected to the anterior chamber by a 23-gauge needle. The IOP levels were maintained for 60 min, and the glutamate levels were determined every 10 min during the 60 min. The mean basal levels of glutamate in the 10-min dialysate were not significantly different among the three groups. The glutamate levels remained unchanged and stable in the controls, but elevation of the IOP significantly increased the level of the glutamate in the dialysate (IOP60, P=0.012; and IOP120, P=0.005: repeated measures ANOVA). Elevation of the IOP causes an increase in the glutamate levels in the ONH of rabbits. This suggests a possible interaction between glutamate metabolism and the IOP in the ONH.