Proteomics is the science that studies the complete set of proteins expressed in a given biological system at a given time. One of its key aims is to measure dynamic changes in protein quantities and posttranslational modifications as a function of physiological, pathological or experimental perturbations. Together with ultrastructural studies, comparative analysis of the protein composition of the organelles defines their identity and their relationships within the cell. Therefore, the exact identity of the phagosome is crucial to understanding its biology. The phagosome is a cytoplasmic organelle composed of a particle of extracellular origin enclosed by a membrane. It is built de novo upon particle uptake and its composition changes over time due to maturation. As early as the first published purification, the question of the phagosome identity was addressed and its lipid composition and enzymatic activities were compared with those of the plasma membrane [1, 2]. The proteomic characterization of phagosomes led to the stepwise identification of over 600 proteins in Drosophila and 800 in the mouse, among a total estimated number of about 1000 proteins [3, 4]. From these efforts have emerged the concept of a virtual model of the phagosome [5]. Groups of proteins can be recognized as functional entities, allowing phylogenetic reconstruction of their acquisition during evolution. The model has been used as a reference for comparisons with physiological [5] or pathogen-induced alterations [6] as well as a predictive tool for genes affecting uptake of bacteria [3]. The predictive power and biological validation of this proteomic-derived model depends both on the mastering of biological variation and on the robustness of the methodology used. On the one hand, the origin of the phagocytic cell, the type of particle and the time of maturation are the main sources of biological variations. Phagocytic cells can be phylogenetically diverse, from protozoan to higher vertebrates. In addition, the particle can be synthetic or natural. Its physical and biological properties impact on the receptors triggered, rate of uptake and subsequent j107 maturation. After engulfment, the process of maturation, which is naturally dedicated to microbe killing and degradation, will progressively change the environment around the particle, making the phagosome a transient and very dynamic organelle. On the other hand, because phagosome isolation from the cell is an obvious prerequisite of proteomic studies, organelle isolation as well as protein separation and identification techniques are known to introduce biases. In view of such complexity, we will discuss how the knowledge of the protein content of the phagosome has evolved and what are the advantages and drawbacks of a virtual phagosome as a comparative tool to study microbe-containing phagosomes or vacuoles (MCVs).