Directed evolution and ancestral sequence reconstruction experiments have provided evidence for the importance of promiscuous activities in the evolution of new protein functions. The apparent ubiquity of promiscuity in proteins has lent weight to the idea that the modern repertoire of specialized proteins has evolved from multi-functional primordial ancestors; however, this hypothesis has not been tested explicitly. Furthermore, little is known about the mechanistic and thermodynamic features of promiscuous protein-ligand interactions or how these features change as protein-ligand interactions become specialized through evolution. We reconstructed and characterized several ancestors of the polar amino acid-binding proteins, which are involved in amino acid uptake in bacteria, to determine whether these ancient proteins were less specialized than their modern counterparts and to investigate mechanisms of promiscuous binding. Characterization of the binding activities of four ancestral amino acid-binding proteins by isothermal titration calorimetry revealed a strong preference for arginine, and weaker affinities for aspartate, glutamate, lysine, histidine and glutamine. Despite the fact that the ancestral proteins predate the divergence of the major bacterial kingdoms, several are more specific than their descendants, contradicting the hypothesis that they would bind an expanded range of amino acids and providing evidence that specialization of this protein family occurred early in its evolutionary history. However, some examples where a promiscuous ancestral activity appeared to have been co-opted by evolution to produce a high-affinity binding protein were evident; for example, the ancestor of the lysine/arginine/ornithine-binding protein and glutamine-binding protein subfamilies had high affinity for arginine and low affinity for glutamine. The crystal structure of this ancestral protein complexed with arginine suggests a key role for Q116 in binding both arginine and glutamine, permitting a hydrogen bonding interaction with the amide group of glutamine but forcing a nearby loop to adopt an unfavourable conformation. Further structural and thermodynamic characterization of the ancestral proteins will enable more detailed understanding of the mechanisms of promiscuous binding in this protein family.