CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewing's sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase-independent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell–cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.