To study the mechanism of syncytium formation, novel syncytia-inducing ecotropic murine retrovirus was used. Our previous result showed that amino acid substitutions at the RBD (receptor binding domain) of envelope glycoprotein contribute to syncytium formation. In this study, we have investigated if this fusion phenomenon could occur with retroviral vectors pseudotyped with the novel syncytia-inducing ecotropic murine leukemia virus Env. We have found that these vectors were not able to mediate virus-to-cell fusion in M. dunni murine cell lines. These findings indicate that syncytia-inducing ecotropic murine leukemia virus is capable of generating syncytia during its replication. There was also no correlation between the level of ecotropic murine leukemia virus receptor (mCAT-1) and the fusogenic effect.