Fluorescence-activated cell sorting, or flow cytometry, has been invaluable in analyzing heterogeneity and isolating cellular subsets of the immune system and other tissues. Flow cytometry can be very useful in studies of neurons and the nervous system, as well. This article describes a method for analyzing and isolating one class of neurons from the central nervous systems of embryonic rats and mice, namely, the motoneurons of the spinal cord. The method uses retrograde labeling with a fluorescent tracer to identify motoneurons in situ, and cell sorting to Isolate the labeled motoneurons after the labeled spinal cords have been dissociated. With this method, millions of living motoneurons can be Isolated with greater than 98% purity.