Epidermal Growth Factor (EGF) induces the production of hydrogen peroxide (H₂O₂), which plays a key role in EGF receptor (EGFR)-mediated tyrosine phosphorylation. When A431 human epidermoid carcinoma cells are stimulated with EGF, there is a transient increase in intracellular reactive oxygen species (ROS), measured using the fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) and laser-scanning confocal microscopy. The primary ROS produced appears to be H₂O₂, as its generation was completely blocked by introducing catalase into the cells via electroporation. Catalase also inhibited EGF-induced tyrosine phosphorylation of various proteins, including the EGFR and phospholipase C-γ1. The production of H₂O₂ depends on the intrinsic tyrosine kinase activity of the EGFR and its autophosphorylation sites. However, EGF failed to induce H₂O₂ generation in cells expressing a kinase-inactive EGFR. In contrast, cells expressing a mutant EGFR with the 126 COOH-terminal amino acids deleted showed normal H₂O₂ generation, suggesting that the kinase activity, but not the autophosphorylation sites, is essential for EGF-induced H₂O₂ formation. H₂O₂ may inhibit protein tyrosine phosphatase (PTPase) activity, which is necessary for EGF-induced tyrosine phosphorylation. The study also shows that H₂O₂ is a small, diffusible molecule that can be rapidly synthesized and destroyed in response to external stimuli, making it a suitable intracellular messenger. The results suggest that H₂O₂ is involved in EGF signaling by mediating tyrosine phosphorylation, which is essential for downstream signaling events. The study also demonstrates that the intrinsic tyrosine kinase activity of the EGFR is required for H₂O₂ generation, but the autophosphorylation sites are not. This was confirmed by examining cell lines expressing wild-type, kinase-negative, and COOH-terminal deletion mutant EGFRs. Only the wild-type and CD-126 mutant EGFRs showed H₂O₂ generation in response to EGF, while the kinase-negative mutant did not. These findings indicate that the kinase activity, but not the autophosphorylation sites, is essential for EGF-induced H₂O₂ generation. The study highlights the role of H₂O₂ in EGF signaling and its potential as a physiological mediator of cellular responses. The findings suggest that H₂O₂ is involved in the activation of signaling pathways, including those mediated by PLC-γ1, and may play a role in the inhibition of PTPase activity, which is necessary for tyrosine phosphorylation. The results also suggest that H₂O₂ is generated in non-phagocytic cells,Epidermal Growth Factor (EGF) induces the production of hydrogen peroxide (H₂O₂), which plays a key role in EGF receptor (EGFR)-mediated tyrosine phosphorylation. When A431 human epidermoid carcinoma cells are stimulated with EGF, there is a transient increase in intracellular reactive oxygen species (ROS), measured using the fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) and laser-scanning confocal microscopy. The primary ROS produced appears to be H₂O₂, as its generation was completely blocked by introducing catalase into the cells via electroporation. Catalase also inhibited EGF-induced tyrosine phosphorylation of various proteins, including the EGFR and phospholipase C-γ1. The production of H₂O₂ depends on the intrinsic tyrosine kinase activity of the EGFR and its autophosphorylation sites. However, EGF failed to induce H₂O₂ generation in cells expressing a kinase-inactive EGFR. In contrast, cells expressing a mutant EGFR with the 126 COOH-terminal amino acids deleted showed normal H₂O₂ generation, suggesting that the kinase activity, but not the autophosphorylation sites, is essential for EGF-induced H₂O₂ formation. H₂O₂ may inhibit protein tyrosine phosphatase (PTPase) activity, which is necessary for EGF-induced tyrosine phosphorylation. The study also shows that H₂O₂ is a small, diffusible molecule that can be rapidly synthesized and destroyed in response to external stimuli, making it a suitable intracellular messenger. The results suggest that H₂O₂ is involved in EGF signaling by mediating tyrosine phosphorylation, which is essential for downstream signaling events. The study also demonstrates that the intrinsic tyrosine kinase activity of the EGFR is required for H₂O₂ generation, but the autophosphorylation sites are not. This was confirmed by examining cell lines expressing wild-type, kinase-negative, and COOH-terminal deletion mutant EGFRs. Only the wild-type and CD-126 mutant EGFRs showed H₂O₂ generation in response to EGF, while the kinase-negative mutant did not. These findings indicate that the kinase activity, but not the autophosphorylation sites, is essential for EGF-induced H₂O₂ generation. The study highlights the role of H₂O₂ in EGF signaling and its potential as a physiological mediator of cellular responses. The findings suggest that H₂O₂ is involved in the activation of signaling pathways, including those mediated by PLC-γ1, and may play a role in the inhibition of PTPase activity, which is necessary for tyrosine phosphorylation. The results also suggest that H₂O₂ is generated in non-phagocytic cells,