Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (Gagnon, Duclos et al. 2002). To reconcile these disparate hypotheses, we used a combination of biochemical, fluorescence imaging and electron microscopy techniques to quantitatively and dynamically assess the contribution of the plasmalemma and ER during phagocytosis. We could not verify even a transient physical continuity between the ER and the plasma membrane, nor could we detect a significant contribution of the ER to forming or maturing phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main constituent of nascent and newly formed phagosomes, which are progressively remodeled by fusion with endosomal and lysosomal constituents as phagosomes mature into acidic, degradative organelles.