OBJECTIVE Ethambutol can cause optic neuropathy and deficiencies in color-opponent visual processing in patients treated for tuberculosis. In fish, Ethambutol induces color vision deficiencies similar to those observed in humans and affects color coding in retinal ganglion cells. Color opponency in fish is mainly mediated by a horizontal cell feedback onto cones thought to be provided by spinules. The authors examined whether Ethambutol affects spinules and is, therefore, able to alter color processing at a distal stage, that is, at the first synaptic connection within the retina. METHODS Ethambutol was injected into the vitreous of either dark- or light-adapted fish. After drug application, fish were held under different illumination conditions. Thereafter, the retinas were dissected and prepared for electron microscopy. Ultrathin tangential sections of retinas were examined at the level of the outer plexiform layer. RESULTS In already light-adapted retinas, a high dose of Ethambutol (10 mM) reduced the number of spinules by 30%. Ethambutol application in the dark with subsequent light adaptation resulted in severe dose-related inhibition of light-induced spinule formation. In these experiments, low doses (0.1 mM) of Ethambutol caused 40% inhibition, and high doses (10 mM) caused 70% inhibition. Besides affecting spinules, Ethambutol occasionally induced a degeneration of cone pedicles. This neurotoxicity only occurred in cones exposed to light. CONCLUSIONS Results show that Ethambutol alters synaptic connections between horizontal cells and cones in a dose-related fashion; Ethambutol treatment can be toxic for cone pedicles and can cause their degeneration; and the rod pathway is not affected by the drug. This indicates that Ethambutol influences the color-coding process already at the level of the cone-horizontal cell synapse.