G protein-coupled receptors (GPCRs) are integral membrane proteins involved in cellular signaling and constitute major drug targets. Despite their importance, the relationship between structure and function of these receptors is not well understood. In this study, the role of extracellular disulfide bonds on the trafficking and ligand-binding activity of the human A2A adenosine receptor was examined. To this end, cysteine-to-alanine mutations were conducted to replace individual and both cysteines in three disulfide bonds present in the first two extracellular loops. Although none of the disulfide bonds were essential for the formation of plasma membrane-localized active GPCR, loss of the disulfide bonds led to changes in the distribution of the receptor within the cell and changes in the ligand-binding affinity. These results indicate that in contrast to many class A GPCRs, the extracellular disulfide bonds of the A2A receptor are not essential, but can modulate the ligand-binding activity, by either changing the conformation of the extracellular loops or perturbing the interactions of the transmembrane domains.