Influence of H 2 O 2 on glycolysis was investigated. A hypothesis previously formulated was tested according to which a mild oxidation of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) results in uncoupling of oxidation and phosphorylation at this step of glycolysis due to acylphosphatase activity of the oxidized enzyme. Incubation of a mixture of purified glycolytic enzymes, as well as a muscle extract, in the presence of 10‐100 μMH 2 O 2 was shown to result in an increase in the rate of glycolysis. The level of lactate accumulation in the oxidized samples increased by 80‐150% compared to the samples containing mercaptoethanol. No ATP was formed by the H 2 O 2 ‐stimulated glycolysis. Thus, H 2 O 2 really caused uncoupling of oxidation and phosphorylation in glycolysis. A role of GAPDH oxidation in regulation of glycolysis is discussed.