In this study, double immunofluorescence methods were used to investigate possible colocalization of the neurotransmitters dopamine [DA] and GABA in rostral spinal cord neurones in the upstream migrating adult sea lamprey (Petromyzon marinus). Double immunofluorescence revealed that all the DA-immunoreactive (ir) cerebrospinal fluid-contacting (CSF-c) cells, approximately 30% of the medioventral DA-ir cells, and most of the DA-ir cells located in the grey lateral to the central canal were also GABA-ir. The results also revealed some DA-ir cells located dorsally to the central canal, which increases the number of dopaminergic cell types known in lamprey. Double-labelled fibres were mainly distributed in the ventral column, and double-labelled boutons contacted some dorsal GABA-ir CSF-c cells, as well as some non-CSF-c GABA-ir cells and ventromedial dendrites of motoneurones. The findings reveal colocalization of dopamine and GABA in some cells and fibres, which suggests co-release of these substances in some synaptic terminals. Although dopaminergic/GABAergic CSF-c cells have been reported in some other vertebrates, the other double-labelled spinal populations appear exclusive to lampreys.