In order to obtain markers selective for motor neurons, an in vitro immunization was carried out using a crude homogenate of embryonic rat ventral spinal cord. We have generated a monoclonal antibody, MO-1, that binds selectively to the cell bodies of somatic motor neurons in the brain stem and spinal cord of the adult rat nervous system. In a survey of both peripheral and central nervous systems, intense labeling by MO-1 appears exclusive to this class of cholinergic neuron. Immunoreactivity is predominantly intracellular and is detectable within the somata as well as the proximal regions of processes but is absent along fiber tracts and at neuromuscular junctions. This staining pattern indicates that MO-1 does not recognize other molecules known to be present in motor neurons, such as choline acetyltransferase, acetylcholinesterase, agrin, or the calcitonin-gene-related-peptide. In the spinal cord, antibody binding begins to be detectable in motor neurons late in development, during the second postnatal week. Thus, MO- 1 appears to recognize a novel cellular component that accumulates in somatic motor neurons during terminal stages of differentiation.