The cephalochordate amphioxus is the closest invertebrate relative to vertebrates. In this study, using HPLC technique, free L-amino acids (L-AAs) and D-aspartic acid (D-Asp) have been detected in the nervous system of the amphioxus Branchiostoma lanceolatum. Among other amino acids glutamate, aspartate, glycine, alanine and serine are the amino acids found at the greatest concentrations. As it occurs in the nervous system of other animal phyla, glutamate (L-Glu) and aspartate (L-Asp) are present at very high concentrations in the amphioxus nervous system compared to other amino acids, whereas the concentration of taurine and γ-aminobutyric acid (GABA) is very low. Interestingly, as it is the case in vertebrates, D-aspartic acid is present as an endogenous compound in amphioxus nervous tissues. The physiological function of excitatory amino acids, and D-aspartate in particular, are discussed in terms of evolution of the nervous system under an Evo-fun (Evolution of function) perspective.