CD50 (ICAM-3) has been identified as the third CD11a/CD18 (LFA-1) counter receptor. We investigated the expression and possible role of this molecule in the induction of early and late activation events in human thymocytes. We observed that CD50 expression is acquired by early T cell progenitors (CD34+) and maintained during thymic development, reaching the highest levels in the most mature population of thymocytes (CD3high). Neither basal nor cytokine-induced expression of CD50 was observed on untransformed human thymic epithelial cell lines. Cross-linking of CD50 expressed on the surface of human thymocytes, by using mAbs recognizing epitopes not related to the CD11a binding site, transduced transmembrane signals leading to an increase of intracellular calcium concentration. This calcium mobilization was inhibited when CD50 was co-cross-linked with CD45, suggesting that tyrosine phosphorylation is also involved in CD50 signaling. The same anti-CD50 mAbs that were able to affect intracellular calcium levels were shown to induce CD69 but not CD25 expression on human thymocytes. This effect was preferentially observed on CD3low/CD3high thymocyte subpopulations. Cross-linking of CD50 also significantly increased activation-induced cell death of human thymocytes. These results support the idea that CD50 molecule can play a role in developing functionally mature T lymphocytes.