CD24 is a differentiation antigen expressed by murine hematopoietic and neural cells which is linked to the membrane via a glycosylphosphatidylinositol (GPI) anchor. In monocytic ESb-MP cells the molecule serves as a ligand for P-selectin and triggering with CD24 specific antibodies can activate VLA-5/L1-mediated cell adhesion in these cells. We report here that the aggregation is specific for CD24 and not seen with antibodies to the GPI-anchored molecule Thy-1. The Tyr-kinase inhibitor herbimycin can block the aggregation. We studied CD24 associated molecules that might be involved in signal transduction. Antibodymediated crosslinking of CD24 induced a rapid Tyr-phosphorylation of several cellular proteins in ESb-MP cells which correlated with an elevated activity of p56lck but not p60fyn or MAP-1 kinase. Several phosphorylated proteins were co-immunoprecipitated with CD24. Re-immunoprecipitation allowed the detection of p56lck, p56hck, and p54lyn but not p60fyn, PI-3k, or PLCgamma as a compenent of the CD24 detergent resistant complex. It is suggested that the CD24-associated kinases are involved in the activation of cell aggregation.