Retrograde axonal transport method of the fluorescent tracer FluoroGold (FG) was combined with immunocytochemistry to investigate the occurrence of nitric oxide synthase (NOS), somatostatin (SOM), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) in both intrinsic and cortically projecting neurons of the rat claustrum. Only NOS was detected in both the scattered projecting neurons and internal neurons of the claustrum. Approximately 20% of NOS-immunoreactive neurons in the claustrum were also retrogradely labeled with FG after tracer injections into the frontal cortex. The other substances were exclusively confined to the population of interneurons, which mainly displayed an oval, round or fusiform shape and a medium size. Apart from the neuronal somata, the proximal parts of the dendritic arborization were clearly visible. The immunoreactive neurons were randomly distributed in the claustrum and their neuronal size and shape did not differ in the various parts of the studied structure. Co-localization of NOS and SOM or NOS and NPY was reported. In conclusion, SOM, VIP and NPY do not appear to play a significant role in the claustro-cortical projection but are most probably involved in modulation and information transfer in the claustrum. The appearance of NOS in both cortically projecting and intrinsic neurons of the claustrum may be indicative of a fundamentally different role in the functioning of the claustro-cortical loop.