Prior to the cloning of G protein-coupled receptors (GPCRs), structure-function analysis of the ligand-binding properties of these receptors had for the most part been limited to study of the structure-activity relationships (SAR) of agonists and antagonists at the individual receptors. For a particular receptor, these SAR data were obtained via synthesis of series of chemically modified hgands, followed by the determination of their binding activities in tissues or cells that natively expressed the receptor of interest. With the molecular cloning of a multitude of GPCRs over the last several years, the amino acid sequence of these receptors is now known. With this knowledge, structural features of the receptors that are involved in ligand binding may now be explored in mutagenesis studies. Indeed, a principal focus of receptor research over recent years has been the detailed structure-function analysis of the ligand-binding properties of genetically engineered receptors heterologously expressed in the appropriate cell systems. Through such studtes, receptor regions and even single amino acids involved in ligand recognition have been identified. This chapter details the techniques used in our laboratory for the construction and analysis of receptors possessing single ammo acid point mutations and receptors composed of amino acid sequence derived from two parent wild-type receptors, i.e., chimeric receptors. Specifically, we have used these techniques to study the structural features responsible for the binding properties of different adenosine receptor subtypes (1 –3 ). Similar approaches have been used by many laboratories focusing on many different GPCRs (reviewed in refs. 4 –6 ).