In birds there are segmentally organized marginal nuclei at the lateral or ventrolateral border of the spinal cord. In most regions of the spinal cord these nuclei are within the outline of the cord. However, in the lumbosacral region they form accessory lobes protruding into the vertebral canal. Histochemical and immunocytochemical investigations were performed to study the neurochemical features of the marginal nuclei of the pigeon. Despite histological differences (only accessory lobe neurons are embedded in glia-derived glycogen cells), there was no difference in the chemical neuroanatomy of the two types of marginal nuclei. These nuclei contained cholinergic neurons and there was also evidence for a cholinergic innervation. NADPH-diaphorase activity, which is considered to indicate nitric oxide synthesis, was faint in marginal neurons. No serotonin immunoreactivity was found. However, all neurons showed immunoreactivity to glutamate and glycine, and some were immunoreactive to gamma-aminobutyric acid (GABA). A GABAergic innervation of non-GABAergic neurons could also be demonstrated. The lack of difference in the chemical neuroanatomical features between cervical marginal nuclei and lumbosacral accessory lobes suggests a similar origin of all marginal neurons. A comparison with the chemical neuroanatomy of marginal neurons in other vertebrates shows both similarities and differences.