Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis and some non-apoptotic activation events. It has been proposed that PS externalization is regulated by the activity of phospholipid scramblase 1 (PLSCR1), a Ca²⁺-dependent endofacial plasma membrane protein, which is tyrosine phosphorylated in activated cells. It is however unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization and exocytosis. Using rat basophilic leukemia cells as a model, we show that non-apoptotic PS externalization induced through the high-affinity IgE receptor (FcRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in Fc{epsilon]RI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1, its tyrosine phosphorylation, PS externalization and exocytosis are independent phenomena which could be distinguished by employing specific conditions of activation.