Motor neurons isolated from 6-day-old embryonic chick spinal cords require muscle extract for survival in culture; however, it was found, that some motor neurons, identified by retrograde labeling with rhodamine, will survive in mixed spinal cell cultures in the absence of the extract. The motor neuron survival-promoting activity produced by spinal cells is soluble and differs from the factor present in muscle extract, the two activities acting in a synergistic manner: the spinal cell activity potentiated that of muscle to decrease its ED50 by an order of magnitude, the motor neuronal survival (30%) seen in the presence of both factors being more than the sum of their individual activities. This synergism was shown to be restricted to the action of the spinal cell factor on motor neurons, no effect of the factor being noted with sympathetic neurons. As a series of defined growth and survival factors present in the central nervous system (nerve growth factor, brain-derived neurotrophic factor, acidic and basic fibroblast growth factors) had no effect on motor neuron survival, we conclude that the molecule responsible for the motor neuron survival-promoting activity of the spinal cells is a previously undefined factor.