Reactive oxygen species (ROS) are key components of postreceptor intracellular signaling pathways; however, the role of ROS in signal initiation is uncertain. We discovered that receptor–ligand interaction caused the generation of hydrogen peroxide (H₂O₂). Using members of the hematopoietin receptor superfamily, as well as EGF receptor, we show that H₂O₂ is generated by specific receptor–ligand interaction in cells and in cell-free systems. With cognate ligand, the extracellular domain of the receptor was sufficient for H₂O₂ generation. We also found that production of H₂O₂ was diminished in a granulocyte–macrophage colony-stimulating factor receptor mutant unable to bind ligand. Exogenously added H₂O₂ induced signaling in the absence of ligand, whereas catalase and a membrane-bound peroxiredoxin inhibited ligand-dependent signaling. Our results suggest that H₂O₂ produced by receptor–ligand interaction is involved as a chemical mediator that facilitates cell signaling.