Exposure of rat adipocytes to insulin causes activation of a pyridine nucleotide oxidase at the internal side of the plasma membrane, generating hydrogen peroxide (S. P. Mukherjee and W. S. Lynn, Fedn Proc. 35, 1694 (1976); S. P. Mukherjee and W. S. Lynn, Archs Biochem. Biophys. 184, 69 (1977). Evidence was also presented that intracellular utilization of H2O2 by the peroxidative pathways of glutathione peroxidase and catalase is coupled with glucose oxidation via the pentose phosphate pathway [S. P. Mukherjee, R. H. Lane and W. S. Lynn, Biochem. Pharmac. 27, 2589 (1978)]. The relationship between the glucose-independent insulin effect on H2O2 production and its metabolic role is evaluated on the basis of formate oxidation, lipogenesis, antagonism of lipase activity and lowering of cellular levels of cyclic 3',5'-adenosine monophosphoric acid (cAMP). These measures of the effects of insulin, observed at low concentrations of glucose, were reversed at higher concentrations of glucose (over 0.3 mM). Exogenous H2O2 had metabolic effects similar to insulin. Addition of H2O2 (10-4 M and higher) to the extracellular medium caused a substantial inhibition of epinephrine-stimulated and adrenocorticotropin-stimulated depot-fat lipase activity in these cells, measured by net glycerol production. H2O2 also increased lipogenesis by increasing the provision of the substrates and cofactor (NADPH) and activating pyruvate dehydrogenase in the same manner as insulin. Exogenous catalase (16 μg/ml) abolished the insulin-like effects of H2O2 (but not of insulin itself) on glucose oxidation, the contribution of glucose carbons to glyceride fatty acids and glyceride glycerol, the inhibition of lipolysis, and the time-dependent decline in the cAMP content of the cells. The data suggest that, while insulin-stimulated H2O2 production from the plasma membrane may bring about some glucose-independent metabolic effects through a lowering of cytoplasmic redox potential, increasing availability of d-glucose may counterbalance these effects by replenishing the reducing equivalents. Endogenous H2O2 appears, therefore, to satisfy the manifold criteria for a 'second messenger' of insulin. © 1980.