In order to investigate the requirement for lateral mobilization of viral envelope glycoproteins on the cell surface in the induction of cell-cell fusion, we employed fluorescence photobleaching recovery to study the effect of the fusion temperature on the lateral mobilization of Sendai virus glycoproteins in the human erythrocyte membrane. As the fusion temperature was reduced below 37 °C (to 31 or 25 °C), the rates of virus-cell fusion, the accompanying hemolysis, and cell-cell fusion were all slowed down. However, the plateau (final level) after the completion of fusion was significantly reduced at lower fusion temperatures only in the case of cell-cell fusion, despite the rather similar final levels of virus-cell fusion. A concomitant decrease as a function of the fusion temperature was observed in the fraction of cell-associated viral glycoproteins that became laterally mobile in the erythrocyte membrane during fusion, and a strict correlation was found between the level of laterally mobile viral glycoproteins in the cell membrane and the final extent of cell-cell fusion. The accompanying reduction in the lateral diffusion coefficients (D) of the viral glycoproteins (1.4-fold at 31 °C and 1.9-fold at 25 °C, as compared to 37 °C) does not appear to determine the final level of cell-cell fusion, since fusing the cells with a higher amount of virions at 25 °C increased the final level of cell-cell fusion while D remained constant. The results demonstrate that lateral mobilization of the viral glycoproteins in the target cell membrane is not an immediate consequence of viral envelope-cell fusion and support the view that it plays an essential role in the induction of cell-cell fusion by native Sendai virions.