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The development, morphological and neurochemical properties of specific populations of interneurons were investigated in the ventral horn of the embryonic and mature chick lumbosacral spinal cord by using pre- and post-embedding immunocytochemical as well as anterograde axonal tracing techniques. We have identified and traced the morphological maturation of the following cell groups: (1) Neurons immunoreactive for calbindin-D 28k (CaB), a calcium-binding protein that has been reported to be a marker of certain subsets of excitatory spinal neurons. We have distinguished and traced the maturation of three CaB-immunoreactive cell groups in the ventral horn; (2) Neurons immunoreactive for GABA and glycine, the two putative inhibitory amino acid neurotransmitters in the spinal cord; (3) Neurons within the nucleus marginalis, a cell group located in the ventrolateral aspect of the white matter in close proximity to the lateral motor column. The characteristic features of the development of these neurons are discussed and correlated with previous neuroanatomical and physiological studies concerning motor functions in the developing chick spinal cord.