The excitatory amino acid l-aspartate (Asp) plays a number of roles in neuronal function. We studied the distribution of Asp-immunoreactive (ir) cells in the telencephalon of young and upstream migrating adult sea lamprey, Petromyzon marinus, and compared it with the distribution of gamma-aminobutyric acid (GABA) immunoreactivity, by using double immunofluorescence methods. Our results reveal for the first time the existence of Asp-ir neuronal populations in the lamprey forebrain. In the olfactory bulbs, Asp-ir neurons were observed in the mitral cell layer and in the inner cellular layer. Many granule-like cells were both Asp-ir and GABA-ir. In the pallium, Asp-ir cells were abundant in the lateral pallium and most of them were also GABA-ir. In the septum/terminal lamina nucleus, some cerebrospinal fluid-contacting type (CSF-c) cells were either Asp-ir or GABA-ir, and a few were double-labeled. Some non-CSF-c septal cells were both Asp-ir and GABA-ir. In the striatum, Asp-ir and/or GABA-ir cells were either subependymal or located in the characteristic arched cell row. In the lateral preoptic region, a few small Asp-ir/GABA-ir neurons were observed. In the caudal preoptic recess nucleus, numerous CSF-c cells were Asp-ir and/or GABA-ir. This study also reveals that colocalization of GABA and Asp immunoreactivities in telencephalic neurons is partial. Further investigation is required to establish whether Asp is a neurotransmitter and/or an intermediate in GABA synthesis in lamprey telencephalon.