Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, CD31) is a 130-kd member of the immunoglobulin gene superfamily that is expressed on the surface of platelets, endothelial cells, myeloid cells, and certain lymphocyte subsets. PECAM-1 has recently been shown to contain functional immunoreceptor tyrosine-based inhibitory motifs (ITIMs) within its cytoplasmic domain, and co-ligation of PECAM-1 with the T-cell antigen receptor (TCR) results in tyrosine phosphorylation of PECAM-1, recruitment of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2), and attenuation of TCR-mediated cellular signaling. To determine the molecular basis of PECAM-1 inhibitory signaling in lymphocytes, the study sought to (1) establish the importance of the PECAM-1 ITIMs for its inhibitory activity, (2) determine the relative importance of SHP-2 versus SHP-1 in mediating the inhibitory effect of PECAM-1, and (3) identify the protein tyrosine kinases required for PECAM-1 tyrosine phosphorylation in T cells. Co-ligation of wild-type PECAM-1 with the B-cell antigen receptor expressed on chicken DT40 B cells resulted in a marked reduction of calcium mobilization-similar to previous observations in T cells. In contrast, co-ligation of an ITIM-less form of PECAM-1 had no inhibitory effect. Furthermore, wild-type PECAM-1 was unable to attenuate calcium mobilization in SHP-2-deficient DT40 variants despite abundant levels of SHP-1 in these cells. Finally, PECAM-1 failed to become tyrosine phosphorylated in p56(lck)-deficient Jurkat T cells. Together, these data provide important insights into the molecular requirements for PECAM-1 regulation of antigen receptor signaling.