CD31 or platelet/endothelial cell adhesion molecule (PECAM‐1) is a 130‐kDa glycoprotein expressed on endothelial cells, granulocytes, a subset of lymphocytes and platelets. In this study, we examined the ability of four monoclonal antibodies (mAb) against different domains of CD31 to modulate the function of T lymphocytes, monocytes and neutrophils. Engagement of CD31 on T lymphocytes results in co‐stimulation of T lymphocyte proliferation to suboptimal doses of anti‐CD31 mAb. This proliferation is accompanied by secretion of numerous cytokines and chemokines, up‐regulation of CD25 and an increase in cell size. Purification of T lymphocytes into CD45RO and CD45RA subsets showed that only naive CD45RA T lymphocytes are co‐stimulated by anti‐CD31 mAb. Further studies on neutrophils show that engagement of CD31 results in down‐regulation of CD62L and up‐regulation of CD11b/CD18 as well as oxidative burst, as assessed by superoxide release. In addition, ligation of CD31 on monocytes results in TNF‐α secretion, and studies with various cell signaling inhibitors indicate that tyrosine kinases and cAMP‐dependent kinases are involved in monocyte activation via CD31. Of the four mAb used in this study, only two activated human leukocytes. These mAb were PECAM‐1.3 and hec7, which bind to domains 1 and 2 of CD31. We conclude that engagement of domains 1 and 2 of CD31 results in outside‐in signaling in leukocytes.