Summary—The fluorescent hydrophobic plasma membrane probe, trimethylamino‐diphenylhexatriene (TMA‐DPH) was previously shown to follow the plasma membrane throughout its internalization and recycling process and thus to behave as a marker for endo‐ and exocytosis in living cell systems. In this paper, we made use of these properties to investigate membrane fluidity effects associated with endocytosis in L929 cells. For that purpose we performed TMA‐DPH fluorescence anisotrophy measurements which showed that endocytosis starts from particularly rigid regions of the plasma membrane (probably coated pits). The fluorescence anisotropy then continuously decreases to a lower limit corresponding to the membrane fluidity of the probe in the lysosomial membrane. Strikingly, the value of this limit is identical to the average anisotropy value in the peripheral membrane, which suggests that lysosomes and plasma membrane may have a similar phospholipidic composition and a possible comm