CD50, the intercellular adhesion molecule-3 (ICAM-3), is expressed almost exclusively on hematopoietic cells. T lymphocytes display a bimodal distribution on CD50 expression levels. It was observed that CD45RO+ cells expressed higher levels of CD50 than CD45RA+ T lymphocytes. A similar situation was observed when CD4 and CD8 subpopulations were analyzed, with CD8+ cells expressing higher levels of CD50 than CD4+ cells. When adult T lymphocytes were analyzed by three-color flow cytometry in CD8+CD45RA+ cells both CD50low and CD50high expressing cells were detected, in accordance with several memory markers on T lymphocytes, whereas only cells with a low level of CD50 were observed in CD4+CD45RA+. The different level of CD50 expression was confirmed by analyzing purified CD45RA+ and CD45RO+ T cells. Moreover, after the comparison of CD50 expression level in thymocytes, cord blood and adult T lymphocytes, a progressive increase was observed. When T cells were sorted by their intensity of CD50 expression, only CD50high cells proliferated in response to tetanus toxoid. Therefore, the phenotypic and functional analysis of adult and cord blood T lymphocytes as well as thymocytes indicates that CD50 expression increases during the maturation process of T lymphocytes: from the lowest CD50 levels present on CD1+ thymocytes, to the highest levels of CD50 on human memory T cells. In addition, we have observed that after CD50 cross-linking on human T lymphocytes, a transient increase in intracellular calcium concentration ([Ca2+]i) is produced. When CD45RA+ and CD45RO+ T cells were analyzed, in spite of the level of CD50 expression, the stimulation through CD50 induced a similar level of Ca2+ mobilization in both subpopulations, contrasting with the higher rise in [Ca2+]i induced by CD3 stimulation on CD45RA+ versus CD45RO+. These data suggest that the signal transduction pathways activated after CD50 cross-linking are, at least partially, independent of those involved after CD3 stimulation.