CD31 (PECAM-1) is an immunoglobulin gene superfamily cell adhesion molecule found on vascular endothelium, platelets, and leukocytes. Lymphocyte expression of CD31 is most closely associated with the CD45RA+CD8+ naive T phenotype. CD31 has recently been shown to play a role in leukocyte egress to inflammatory sites. The mechanism of CD31 adhesion remains under investigation. Several investigators have reported evidence for a heterotypic ligand. We have previously shown that CD31 is phosphorylated with cell activation, which suggests a possible role for CD31 in cell activation events. We therefore studied the effects of CD31 antibodies on in vitro assays of lymphocyte activation. One CD31 antibody, LYP21, inhibited the mixed lymphocyte reaction (MLR) in a specific and dose-dependent fashion. An LYP21 epitope was localized to the sixth Ig domain of CD31. This peptide and a scrambled control peptide were synthesized and used to study effects of this epitope on lymphocyte activation. The CD31 peptide strongly inhibited the MLR. Because CD31 is expressed on both stimulator and responder populations, stimulator peripheral blood leukocytes and responder lymphocyte populations were separately incubated with CD31 peptide or control peptide and then washed before mixing. The CD31 peptide inhibited the MLR equally when either stimulator or responder cells were preincubated with the CD31 peptide. We further sorted responder cells into CD31-high and CD31-low populations and separately incubated these subsets with peptides. The CD31 peptide strongly inhibited MLRs, regardless of level of responder-cell CD31 expression. Examination of MLR reactions involving the CD31 peptide showed dispersed small aggregates of cells, rather than the single large aggregate observed in control MLRs. The CD31 peptide did not affect activation of lymphocytes by phorbol myristate acetate (PMA) and ionomycin. These results suggest that a surface CD31-ligand interaction may have a functional role in alloimmune lymphocyte activation and identify a functionally important domain of CD31.