Hydrogen peroxide (H 2O 2) released by neutrophils is an important mediator of endothelial cell (EC) injury and vascular inflammation via its effect on EC-free Ca 2+, [Ca 2+] i. Although the underlying mechanisms are not well understood, platelet endothelial cell adhesion molecule (PECAM)-1/CD-31 is a critical modulator of neutrophil–EC transmigration. PECAM-1 is also known to regulate EC calcium signals and to undergo selective tyrosine phosphorylation. Here, we report that PECAM-1 molecules transduce EC responses to hydrogen peroxide. In human umbilical vein EC and REN cells (a PECAM-1–negative EC-like cell line) stably transfected with PECAM-1 (RHP), noncytolytic H 2O 2 exposure (100–200 μM H 2O 2) activated a calcium-permeant, nonselective cation current, and a transient rise in [Ca 2+] i of similar time course. Neither response was observed in untransfected REN cells, and H 2O 2-evoked cation current was ablated in REN cells transfected with PECAM-1 constructs mutated in the cytoplasmic tyrosine–containing domain. The PECAM-dependent H 2O 2 current was inhibited by dialysis of anti–PECAM-1 cytoplasmic domain antibodies, required Src family tyrosine kinase activity, was independent of inositol trisphosphate receptor activation, and required only an intact PECAM-1 cytoplasmic domain. PECAM-1–dependent H 2O 2 currents and associated [Ca 2+] i transients may play a significant role in regulating neutrophil–endothelial interaction, as well as in oxidant-mediated endothelial response and injury.