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Chemotaxis is the primary mechanism by which cell movements are directed within multicellular organisms, and it is a major component of embryonic development, wound healing, and immune responses. Chemotaxis involves a complex cascade of events--formation of signaling complexes, receptor polarization, adhesion molecule activation, and cytoskeletal reorganization. Previous assay methods were limited in several ways that reduced users' abilities to obtain quantitative data or to control conditions precisely. We describe a unique chemotactic assay that can incorporate multiple chemotactic gradients in different spatial and temporal combinations. In addition, this assay is easily adapted for live-cell imaging and fluorescent microscopy. With its relative simplicity, flexibility, and precision, this method is a key tool for the study of cellular chemotactic responses and the signaling processes underlying them.