The topographic distribution of calretinin-immunoreactive neurons was studied in the medial septum diagonal band of Broca complex of the rat, in relation to the localization of other neurochemically identified cell groups containing choline acetyltransferase, parvalbumin or calbindin D28k. Double-labelling experiments revealed that these four antigen-containing cells formed distinct dorsoventrally running lamellae overlayed on top of each other similar to onion leaves. There was only a slight overlapping of the various cell groups. None of the four antigens were co-localized in the same cells. The lamella occupied by calretinin-positive neurons is situated at the border of the medial septum and the intermediolateral septal nucleus, and shows some overlap with the area occupied by cholinergic neurons. Retrograde transport of horseradish peroxidase from the hippocampus combined with immunostaining for calretinin revealed that calretinin-containing neurons do not participate in the septohippocampal projection. The lack of projection to the amygdala was also confirmed. Thus, calretinin-containing neurons represent a distinct cell group in the medial septal region, which either projects to subcortical areas, or may function as interneurons relaying hippocampal feedback to the medial septal projection neurons.