We investigate whether the fourth intracellular loop (IL4) of D1 and D5 dopaminergic receptors (D1R, D5R) confers D1-like subtype-specific signaling properties. Using chimeric receptors (D1R-IL4B and D5R-IL4A), we show that swapping of IL4 leads to a switch in dopamine affinity and constitutive activity of D1R and D5R. Dopamine potency was reduced for both chimeras in comparison with wild-type receptors. Moreover, dopamine-mediated maximal activation was drastically increased in cells expressing D1R-IL4B when compared with those harboring D5R-IL4A or wild-type receptors. In conclusion, IL4 plays a pivotal role in imparting subtype-specific ligand binding and activation properties to highly homologous seven-transmembrane receptors.