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Glutamate and γ-aminobutyric acid (GABA) are major excitatory and inhibitory retinal neurotransmitters. The balance between these signals is a key principle of organization at retinal level. Although glutamate-induced excitotoxicity could mediate retinal ganglion cell death in glaucoma, the GABAergic system was not previously examined in this disease. The aim of this work was to study the retinal GABAergic activity in eyes with ocular hypertension induced by hyaluronic acid (HA). For this purpose, weekly injections of HA were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with saline solution. At 3 weeks of treatment with HA, GABA turnover rate, glutamic acid decarboxylase activity, and both glutamate- and high K
+-induced GABA release significantly decreased, whereas GABA uptake increased in HA-treated eyes. The binding of
t-butylbicyclophosphorothionate (TBPS) to GABA
A/benzodiazepine Cl
− channels significantly increased in eyes injected with HA as compared with vehicle-injected eyes. Changes in GABA uptake and TBPS binding persisted at 6 weeks of treatment with HA. These results indicate a dysfunction of the retinal GABAergic activity in hypertensive eyes, which could suggest the involvement of GABA in glaucomatous neuropathy.