We investigated the expression of nitric oxide synthase (NOS) isoforms -1, -2 and -3 in the retina and optic nerve head (ONH) in an experimental rat model of elevated intraocular pressure (IOP) before and after treatment with timolol, to assess whether its neuroprotective action is associated with the activity of these enzymes. Episcleral vein cauterization in unilateral eyes of Wistar rats was performed to produce elevated IOP. Histological sections of retina and ONH from animals with normal IOP, with elevated IOP, and elevated IOP treated with timolol, were studied by immunohistochemistry with antibodies to NOS-1, NOS-2, and NOS-3. In the control rats, NOS-1 was localized to photoreceptor inner segments, amacrine cells and bipolar cells in the retina, and in astrocytes, pericytes and vascular nitrergic terminals in the ONH. NOS-3 immunostaining localized to the endothelial cells. The rats with elevated IOP showed increased expression of NOS-1 in the plexiform layers of the retina and reactive astrocytes in the ONH. These cells also showed NOS-2 positivity. The rats treated with timolol showed reduced expression of NOS-1 in the retina and ONH. NOS-2 was only detected in a few groups of astrocytes in the ONH. NOS-3 was unchanged in both elevated IOP and timolol-treated groups. These results show that excessive levels of NO synthesized by the NOS-1 and -2 isoforms, considered neurotoxic, might contribute to the progressive lesions of retinal ganglion cell axons. Their reduction after treatment suggests a possible neuroprotective effect of timolol in neurons exposed to excessive amounts of NO.