Ouabain is both a cardiac glycoside used in therapy of congestive heart failure and an endogenous steroid hormone. It specifically binds to Na+, K+-ATPase (NKA) and blocks its activity. Overdose of ouabain induces retinal damage. In different species ouabain-induced retinal degeneration affects different cell types. In fish and rabbit ouabain induces retinal cell death preferentially in the ganglion cell layer and outer photoreceptor segments respectively. In rats, the pattern of NKA expression has been studied with most detail among retinal neurons. In addition, ouabain selectively destroyed some types of neurons in rodents. However, ouabain-sensitive retinal neurons remain unclear in rats. We show here that injection of ouabain into the rat vitreous body induced dramatic cell death in the inner nuclear layer (INL). The cell death was time- and dose-dependent. Ouabain-induced dying cells in the INL were TUNEL-positive. Immunohistochemistry analysis revealed that there was a significant decrease in the number of calbindin D-28K- and syntaxin-1-positive horizontal and amacrine cells in the INL of ouabain-treated rat retinas. Thus our results revealed that the horizontal and amacrine cells are the most sensitive cell types to ouabain in the retina of Sprague-Dawley rat.