Insulin receptor‐substrate‐1 (IRS‐1) is a docking protein for several tyrosine kinase receptors. Upon tyrosine phosphorylation, IRS‐1 binds to signaling molecules that express Src homology 2 (SH‐2) binding domains, including phosphatidylinositol 3‐kinase (PI 3‐kinase), phosphotyrosine phosphatase SHP‐2 (Syp), Nck, Crk and Grb‐2. Hydrogen peroxide (H₂O₂) induces tyrosine phosphorylation of key signaling mediators presumably by inhibition of tyrosine phosphatases. In many cell types, the activation of extracellular signal‐related kinases (e.g. MAPK) and other protein kinases by H₂O₂ leads to transcriptional activation. In the current study, we examined the effect of H₂O₂ on IRS‐1 tyrosine phosphorylation in primary cultured rat cerebellar granule neurons. H₂O₂ stimulated the rapid tyrosine phosphorylation of IRS‐1 and p42/p44 MAP kinase, and induced its association with PI 3‐kinase. H₂O₂‐induced IRS‐1 phosphorylation was rapidly reversible (5 min) whereas MAPK phosphorylation persisted for up to 1 h. NMDA reversed H₂O₂‐mediated tyrosine phosphorylation of IRS‐1 and its association with PI 3‐kinase. The dephosphorylation of IRS‐1 by NMDA was calcium‐dependent and was inhibited by the calcineurin inhibitor cyclosporine. Calmodulin‐dependent tyrosine phosphatase activity of calcineurin was observed in vitro using both immunoprecipitated and recombinant tyrosine‐phosphorylated IRS‐1 as substrates. These data highlight the role of multiple phosphatases in the regulation of IRS‐1 tyrosine phosphorylation and identify a novel functional property of calcineurin.