The rotational mobility of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase) was determined by using erythrosin (Er)-labeled monovalent Fab’ antibody fragments directed specifically against either F or HN. By use of time-resolved phosphorescence anisotropy, the rotational mobility of Er-Fab’-viral glycoprotein complexes was studied both in the envelopes of unfused virions bound to erythrocyte ghosts and in the target cell membrane after fusion had occurred. The rotational correlation times (φ) of Er-Fab’-labeled F and HN were rather similar in the envelopes of bound unfused virions, but highly different in membranes of fused cells. The different φ values indicate that F and HN diffuse separately in the target cell membrane and for the major part are not complexed together. The temperature dependence of the φ values of the Er-Fab’-viral glycoprotein complexes revealed a breakpoint at 22 °C for the F proteins both in bound virions and in the membranes of fused cells, and for the HN proteins in the envelopes of bound virions. In all these cases, the φ values increased between 4 and 22 °C, demonstrating a reduction in the rate of rotational diffusion. Further elevation of the temperature reversed the direction of the change in φ. This phenomenon may reflect a temperature-dependent microaggregation of F and HN saturating at ca. 22 °C and presumably related to the fusion mechanism since the breakpoint temperature correlates closely with the threshold temperature for virus-cell and cell-cell fusion. Both of the latter reactions proceed at negligible rates at about 20 °C. Such an interpretation is also in accordance with the temperature dependence of the lateral diffusion of F and HN in the target cell membrane after fusion. The latter measurements demonstrate that the fractions of laterally mobile F and HN in the target cell membrane increase with temperature up to 22 °C but level off at higher temperatures, a finding consistent with the notion that lateral mobility of the two envelope proteins in the target cell membrane is required for the induction of cell-cell fusion.