This chapter deals with the binding of ligands to G protein-coupled receptors, and has a “top down” structure. It begins with basic theory, outlining the fundamental concepts of ligand-receptor interaction, and progressing to a consideration of the binding of agonists and allosterically acting agents. The chapter considers some practical aspects and pitfalls of binding studies, such as buffer composition, incubation time, and radioligand depletion. It describes five different types of binding assay: radioligand dissociation, radioligand association, radioligand saturation, inhibition of radioligand binding by an unlabeled competitor, and interactions of an allosterically acting ligand with both a radioligand and an unlabeled competitor. The chapter provides detailed protocols for culturing Chinese hamster ovary cells expressing muscarinic receptors, preparation of membranes and their characterization with respect to protein and receptor concentration, and a screening assay for allosteric effects on unlabeled acetylcholine binding.