CD44 is a transmembrane glycoprotein involved in various cell adhesion events, including lymphocyte migration, early hemopoiesis, and tumor metastasis. To examine the requirements of CD44 for signal delivery through the extracellular domain, we constructed a chimeric CD44 protein fused to the intracellular domain of Fas on its C-terminus. In cells expressing the CD44-Fas fusion protein, apoptosis could be induced by treatment with certain anti-CD44 mAbs alone, especially those recognizing the epitope group d, which has been previously shown to play a role in ligand binding, indicating that ligation of a specific region of the CD44 extracellular domain results in signal delivery. Of note was that appropriate ligation of the epitope h also resulted in the generation of apoptotic signal, although this region was not thought to be involved in ligand binding. In contrast, the so-called blocking anti-CD44 mAbs (epitope group f) that can abrogate the binding of hyaluronate (HA) failed to induce apoptosis even after further cross-linking with the secondary Ab, indicating that a mere mAb-induced oligomerization of the chimeric proteins is insufficient for signal generation. However, these blocking mAbs were instead capable of inhibiting apoptosis induced by nonblocking mAb (epitope group h). Furthermore, a chimeric protein bearing a mutation in the HA binding domain and hence lacking the ability to recognize HA was incapable of mediating the mAb-induced apoptosis, suggesting that the functional integrity of the HA binding domain is crucial to the signal generation in CD44.