In order to identify early events in the differentiation of motor neurons, the expression of several developmentally regulated, neuronal molecules was investigated by immunohistochemistry on consecutive sections of cervical spinal cord. Motor neurons are among the first neurons to be born and to differentiate within the embryonic rat spinal cord. They undergo their terminal mitosis on embryonic days 10 and 11 (E10-11) and acquire detectable levels of the transmitter synthesizing enzyme, choline acetyltransferase, by E11.5. Staining with antibodies to the 68 kD neurofilament protein revealed motor neurons extending processes out the ventral root as early as E10.5. Monoclonal antibodies to two different epitopes on the cell adhesive molecule, NCAM, bound to myotomes on E10.5, and began to recognize ventral horn neurons by E11. Two other markers of developing neurons, the growth-associated protein, GAP-43, and the surface glycoprotein, TAG-1, were clearly detected on young motor neurons by E11.5. Thus, during the 36 hours following the final mitosis of their precursors, motor neurons acquire cytoskeletal, enzymatic, and cell surface components that distinguish them from other developing cells within the spinal cord. Not all of the newly acquired molecules continue to be expressed by motor neurons. Immunoreactivity for TAG-1 was lost by E12.5, followed by a gradual reduction of immunoreactivity for GAP-43 and the highly polysialylated form of NCAM. By E15, only antibodies to choline acetyltransferase (Phelps et al., J. Comp. Neurol. 307:1-10, 1990), and to neurofilaments, selectively stained motor neurons within the embryonic spinal cord. The transient presence of GAP-43, TAG-1, and the embryonic form of NCAM coincides with a period of vigorous axonal growth and declines when motor neurons reach their targets. This report describes the temporal sequence of early stages in the differentiation of the rodent motor neuronal phenotype. Some of these changes may be related to interactions with their synaptic partners.