A simple, rapid assay for measuring radiolabeled ligand binding to purified plasma membranes was developed. In this assay, membrane proteins and ligand are mixed atop a nonmiscible silicone oil (density = 1.029 g/cm9 and incubated to establish equilibrium. The membrane proteins and bound ligand are then rapidly separated (30-60 s) from unbound ligand by centrifugation at 100,OOOg in a Beckman airfuge. A small amount of unbound ligand is contained in the pellet as extramembranous fluid so that the bound and free ligand remain essentially in equilibrium. Thus, this binding assay is suitable for the characterization of low-affinity (& > lo-* M) binding sites with rapid dissociation rate constants. In addition, measurements and comparisons of the binding of the synthetic chemotactic peptide formylnorleucyl-1eucyl-[3H]phenylanine to purified rabbit neutrophil membranes have been made using the silicone oil centrifugation assay and a filtration binding assay. The results of these experiments illustrate the problems and potential errors associated with nonequilibrium binding assays and emphasize the advantages of using the silicone oil centrifugation binding assay.