The highly variable extracellular loops in G protein-coupled receptors (GPCRs) have been implicated in receptor activation, the mechanism of which is poorly understood. In a random mutagenesis screen on the human adenosine A(2B) receptor (A(2B)R) using the MMY24 Saccharomyces cerevisiae strain as a read-out system, we found that two residues in the first extracellular loop, a phenylalanine and an aspartic acid at positions 71 and 74, respectively, are involved in receptor activation. We subsequently performed further site-directed and site-saturation mutagenesis. These experiments revealed that the introduction of mutations at either of the identified positions results in a wide variety of receptor activation profiles, with changes in agonist potency, constitutive activity, and intrinsic activity. Radioligand binding studies showed that the changes in activation were not due to changes in receptor expression. We interpret these data in the light of the recently revealed structure of the adenosine A(2A)R, the closest homologue of the A(2B)R. The two residues are suggested to be vital in maintaining the tertiary structure of a β sheet in the extracellular domain of the A(2B)R. We hypothesize that deterioration of structure in the extracellular domains of GPCRs compromises overall receptor structure with profound consequences for receptor activation and constitutive activity.