Many different cell types including stem cells, cancer cells, and cells of the immune system use cell-to-cell and cell-to-extracellular matrix interactions to communicate and to move in space, behaviors that are crucial to their physiological and pathophysiological properties. While conventionally two-dimensional (2D) cell culture was used to study these cellular phenomena, complex three-dimensional (3D) environments more truthfully reflect the biochemical and biomechanical properties of the extracellular matrix and allow analysis of cellular functions under physiologically relevant conditions. Here, we present protocols to study individual cells as well as multicellular cell aggregates within a 3D collagen type I matrix that is amenable to microscopy-based analysis. This approach facilitates analyses of migratory behaviors, reciprocal cell-cell and cell-ECM interactions as well as subcellular distributions of proteins of interest in physiologically relevant 3D settings.