Phosphorylation of tyrosine residues on platelet–endothelial cell adhesion molecule-1 (PECAM-1), followed by signal trans- 13 duction events, has been described in endothelial cells following exposure to hyperosmotic and fluid shear stress. However, it is 14 unclear whether PECAM-1 functions as a primary mechanosensor in this process. Utilizing a PECAM-1–null EC-like cell line, we 15 examined the importance of cellular localization and the extracellular and transmembrane domains in PECAM-1 phosphorylation 16 responses to mechanical stress. Tyrosine phosphorylation of PECAM-1 was stimulated in response to mechanical stress in null cells 17 transfected either with full length PECAM-1 or with PECAM-1 mutants that do not localize to the lateral cell–cell adhesion site and 18 that do not support homophilic binding between PECAM-1 molecules. Furthermore, null cells transfected with a construct that 19 contains the intact cytoplasmic domain of PECAM-1 fused to the extracellular and transmembrane domains of the interleukin-2 20 receptor also underwent mechanical stress-induced PECAM-1 tyrosine phosphorylation. These findings suggest that mechano- 21 sensitive PECAM-1 may lie downstream of a primary mechanosensor that activates a tyrosine kinase.