BACKGROUND The evolutionary origin of the telencephalon, the most anterior part of the vertebrate brain, remains obscure. Since no obvious counterpart to the telencephalon has yet been identified in invertebrate chordates, it is difficult to trace telencephalic origins. One way to identify homologous brain parts between distantly related animal groups is to focus on the combinatorial expression of conserved regionalisation genes that specify brain regions. RESULTS Here we report the combined expression of conserved transcription factors known to specify the telencephalon in the vertebrates in the chordate amphioxus. Focusing on adult specimens, we detect specific co-expression of these factors in the dorsal part of the anterior brain vesicle, which we refer to as Pars anterodorsalis (PAD). As in vertebrates, expression of the transcription factors FoxG1, Emx and Lhx2/9 overlaps that of Pax4/6 dorsally and of Nkx2.1 ventrally, where we also detect expression of the Hedgehog ligand. This specific pattern of co-expression is not observed prior to metamorphosis. Similar to the vertebrate telencephalon, the amphioxus PAD is characterized by the presence of GABAergic neurons and dorsal accumulations of glutamatergic as well as dopaminergic neurons. We also observe sustained proliferation of neuronal progenitors at the ventricular zone of the amphioxus brain vesicle, as observed in the vertebrate brain. CONCLUSIONS Our findings suggest that the PAD in the adult amphioxus brain vesicle and the vertebrate telencephalon evolved from the same brain precursor region in ancestral chordates, which would imply homology of these structures. Our comparative data also indicate that this ancestral brain already contained GABA-, glutamatergic and dopaminergic neurons, as is characteristic for the olfactory bulb of the vertebrate telencephalon. We further speculate that the telencephalon might have evolved in vertebrates via a heterochronic shift in developmental timing.