The movement of immune cells is an indispensable prerequisite for their function. All essential steps of cellular immunity rely on the ability of cells to migrate and to interact with each other. Although observation of these phenomena in vivo would be the most physiological approach, intravital imaging is technically very demanding and not optimally suited for routine or high-throughput analysis. Any good in vitro experimental system should reflect the inherent three-dimensionality of cell migration and interaction in living tissues. Data generated over the last decade show that important cellular parameters like cell velocity, cell shape, and the physicodynamics of cell—cell interactions closely resemble values observed in vivo when measured in a three-dimensional (3D) collagen matrix assay, featuring a hydrated network of fibers consisting of type I collagen, the major component of the extracellular matrix. In this chapter, we describe in detail the experimental use of the 3D collagen matrix system. We delineate the preparation of immune cells exemplified by bone marrow-derived dendritic cells and antigen specific T-helper cells of the mouse, the build-up and use of the 3D collagen matrix chamber, the procedures of real time fluorescence microscopic analysis of cell migration and cell—cell interaction, as well as data analysis supported by a self-developed software for computerassisted cell tracking.