In vitro binding of skeletal muscle myosin and the erythrocyte cytoskeleton linker protein, band 4.1, was evaluated in a novel small-volume, filtration-based binding assay. The assay equipment consisted of a plastic grid containing several buffer-filled wells into which were placed small nylon screens. Myosin was covalently tethered to an agarose (Sepharose) support and aliquots of this resin were pipetted onto the surface of the submerged nylon screen. Following addition of radiolabeled protein 4.1, and an appropriate incubation period, the myosin-Sepharose beads and bound protein 4.1 were separated by wicking the buffer from beneath the nylon screen with a piece of filter paper. Nylon screens, with adherent resin beads, and the filter paper wicks were then counted to give the amounts of bound and free protein 4.1, respectively. This system proved to be a rapid, simple, and quantitative method for evaluating the behavior of a myosin binding protein under conditions in which free myosin would be prone to assemble into filaments. Moreover, since the assay separates bound and free components within a few seconds, it is well suited for the analysis of low-affinity interactions.