The movement of cells through extracellular matrix (ECM) is a critical component of many normal and pathological processes in vivo. Consequently, efforts to characterize motility-associated interactions between cells and ECM have led to the development of methods to observe and quantify (assay) the movement of cells under simplified conditions in vitro. In this report, we describe a novel method (the bullseye assay) and apparatus for the concentration of cells into small, precisely sized and shaped circular disks (bullseyes) that serve as starting points for migration of cells within ECM. The same apparatus is used to form the bullseyes and position them at the center of flat disks (windows) of gelled collagen that are supported at the edges by rings of nylon mesh. Complete assemblies, each consisting of a bullseye, collagen window and nylon mesh ring, are transferred to tissue culture wells for assay of cell migration either within or on top of the collagen window. Studies of the migratory responses of three different cell types to specific cytokines demonstrated that the bullseye assay was sensitive, rapid to set up, and easy to use. In conjunction with the bullseye assay, we developed a novel annular grayscale method for quantification of cell migration from digital images. The method is easily mastered, is derived from a measurement program in the public domain, is not subjective and is more discriminative than other techniques of measurement.