Methanol is a toxicant that causes systemic and ocular toxicity after acute exposure. The folate‐reduced (FR) rat is an excellent animal model that mimics characteristic human methanol toxic responses. The present study examines the role of the methanol metabolites formaldehyde and formate in the initiation of methanol‐induced retinal toxicity. After a single oral dose of 3.0 g/kg methanol, blood methanol concentrations were not significantly different in FR rats compared with folate‐sufficient (FS) (control) rats. However, FR rats treated with 3.0 g/kg methanol displayed elevated blood (14.6 mM) and vitreous humor (19.5 mM) formate levels and abnormal electroretinograms (loss of fa‐wave) 48 h postdose. FR rats pretreated with disulfiram (DSF) prior to 3.0 g/kg methanol treatment failed to display these symptoms. Formaldehyde was not detected in blood or vitreous humor with or without DSF treatment, suggesting that formate is the toxic metabolite in methanol‐induced retinal toxicity. Additionally, creating a blood formate profile (14.2 mM at 48 h) similar to that observed in methanol‐treated rats by iv infusion of pH‐buffered formate does not alter the electroretinogram as is observed with methanol treatment. These data suggest that intraretinal metabolism of methanol is necessary for the formate‐mediated initiation of methanol‐induced retinal toxicity.