CXCL12-induced and CXCR4-mediated chemotaxis plays a critical role in the trafficking of lymphocytes, which modulates inflammatory and immune responses. The CXCR4 receptor is down-modulated by its ligand CXCL12, which has been shown to inhibit HIV entry into the cells. However, the molecular mechanisms that regulate CXCL12-induced chemotaxis and down-modulation of the CXCR4 receptor are not well known. We have previously shown that CXCL12 induces the tyrosine phosphorylation of the signaling molecule Cbl and its association with the tyrosine phosphatase SHP2. In the present investigation, we have further explored the role of Cbl in CXCR4-mediated chemotaxis and CXCR4 down-modulation. Cbl, a 120 kd protein that contains a tyrosine kinase binding domain (TKB), can function as an adaptor molecule by binding to various proteins. It also contains a RING finger domain and has been shown to negatively regulate signaling by directing the ubiquitination and degradation of signaling molecules. We found that CXCR4 L1.2 cells overexpressing wild-type Cbl or G306E-Cbl (deletion in the TKB domain) showed reduced migration to CXCL12, only 50% of the vector control. Cells overexpressing 70Z-Cbl (deletion in the RING finger domain) showed no difference in migration as compared to the vector control. We next examined CXCR4 expression in these cells by FACS analysis. We found that cells overexpressing wild-type Cbl and G306E-Cbl had approximately 60% CXCR4 receptor expression as compared to the vector control, while the cells overexpressing 70Z-Cbl had no difference in receptor expression. Cells were then internalized by stimulation with CXCL12 or HIV gp120, and after 2 hours approximately 80% of the vector control cells had internalized, as compared to approximately 90% of the cells overexpressing wild-type Cbl and G306E-Cbl, and 60% of the cells overexpressing 70Z-Cbl. Overexpression of wild-type Cbl or G306E-Cbl slightly increased internalization of the CXCR4 receptor, while 70Z-Cbl impeded this process. This indicates that wild-type Cbl enhances internalization, while the G306E mutation in the tyrosine kinase binding domain has no effect on this function of Cbl. Our results suggest that the RING finger domain of Cbl is important for internalization of the CXCR4 receptor. In addition, we observed that there was no difference in the phosphorylation of Erk 1/2 between the vector control cells and those overexpressing wild-type Cbl, 70Z-Cbl, or G306E-Cbl after stimulation with CXCL12, indicating that these changes in migration and receptor expression act in a MAPK-independent pathway. Taken together, these studies suggest that Cbl plays an important role in CXCR4-mediated chemotaxis and the CXCR4 internalization process. These results may lead to better understanding of HIV pathogenesis and inflammatory responses mediated by CXCR4.