We have developed a series of mouse‐mouse neural hybrid cell lines by fusing the aminopterin‐sensitive neuroblastoma N18TG2 with motor neuron‐enriched embryonic day 12–14 spinal cord cells. Of 30 neuroblastoma‐spinal cord (NSC) hybrids displaying a multipolar neuron‐like phenotype, 10 express choline acetyltransferase, and 4 induce twitching in cocultured mouse myotubules. NSC‐19, NSC‐34, and their subclones express additional properties expected of motor neurons, including generation of action potentials, expression of neurofilament triplet proteins, and acetylcholine synthesis, storage, and release. In addition, NSC‐34 cells induce acetylcholine receptor clusters on cocultured myotubes, and undergo a vimentin‐neurofilament switch with maturation in culture, similar to that occurring in neuronal development. NSC cell lines appear to model selected aspects of motor neuron development in an immortalized clonal system. © 1992 Wiley‐Liss, Inc.