In a primary co-culture of spermatogenic and Sertoli cells of the rat, many spermatogenic cells die by apoptosis and are subsequently engulfed by Sertoli cells. We investigated the mechanism of this phagocytosis reaction. Testicular cells from 20-day-old rats were cultured, and spermatogenic cells and Sertoli cells were separated. When the recovered spermatogenic cells were maintained without Sertoli cells, the viability of the cells decreased and they became more susceptible to phagocytosis by Sertoli cells. Phagocytosis was severely impaired when liposomes containing acidic phospholipids, such as phosphatidylserine, phosphatidylinositol, and cardiolipin, were included in the reaction, whereas those consisting of neutral phospholipids showed little effect. Such anionic liposomes were more efficiently engulfed by Sertoli cells than were the other neutral liposomes. Also, the number of spermatogenic cells that exposed phosphatidylserine to the surface increased when cells were maintained in single culture. The results indicate that upon induction of spermatogenic cell apoptosis, phosphatidylserine and probably other acidic phospholipids, which are normally localized in the inner leaflet of the plasma membrane, translocate to the outer leaflet and serve as a signal for phagocytosis by Sertoli cells.