One of the essential properties of mammalian, including sperm, plasma membranes is a stable transversal lipid asymmetry with the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), typically in the inner, cytoplasmic leaflet. The maintenance of this nonrandom lipid distribution is important for the homeostasis of the cell. To clarify the relevance of lipid asymmetry to sperm function, we have studied the localization of PS in boar sperm cell membranes. By using labeled annexin V as a marker for PS and propidium iodide (PI) as a stain for nonviable cells in conjunction with different methods (flow cytometry, fluorescence and electron microscopy), we have assessed the surface exposure of PS in viable cells during sperm genesis, that is, before and during capacitation as well as after acrosome reaction. An approach was set up to address also the presence of PS in the outer acrosome membrane. The results show that PS is localized in the cytoplasmic leaflet of the plasma membrane as well as on the outer acrosome membrane. Our results further indicate the cytoplasmic localization of PS in the postacrosomal region. During capacitation and acrosome reaction of spermatozoa, PS does not become exposed on the outer surface of the viable cells. Only in a subpopulation of PI-positive sperm cells does PS became accessible upon capacitation. The stable cytoplasmic localization of PS in the plasma membrane, as well as in the outer acrosome membrane, is assumed to be essential for a proper genesis of sperm cells during capacitation and acrosome reaction.