The nonphosphorylated neurofilament marker SMI-32 stains motor neurons in spinal cord slices and stains a subset of cultured spinal neurons ["large SMI-32(+) neurons"], which have a morphology consistent with motor neurons identified in vitro: large cell body, long axon, and extensive dendritic arborization. They are found preferentially in ventral spinal cord cultures, providing further evidence that large SMI-32(+) neurons are indeed motor neurons, and SMI-32 staining often colocalizes with established motor neuron markers (including acetylcholine, calcitonin gene-related peptide, and peripherin). Additionally, choline acetyltransferase activity (a frequently used index of the motor neuron population) and peripherin(+) neurons share with large SMI-32(+) neurons an unusual vulnerability to AMPA/kainate receptor-mediated injury. Kainate-induced loss of these motor neuron markers is Ca2+-dependent, which supports a critical role of Ca2+ ions in this injury. Raising extracellular Ca2+ exacerbates injury, whereas removal of extracellular Ca2+ is protective. A basis for this vulnerability is provided by the observation that most peripherin(+) neurons, like large SMI-32(+) neurons, are subject to kainate-stimulated Co2+ uptake, a histochemical stain that identifies neurons possessing Ca2+-permeable AMPA/kainate receptor-gated channels. Finally, of possibly greater relevance to the slow motor neuronal degeneration in diseases, both large SMI-32(+) neurons and peripherin(+) neurons are selectively damaged by prolonged (24 hr) low-level exposures to kainate (10 microM) or to the glutamate reuptake blocker L-trans-pyrrolidine-2,4-dicarboxylic acid (100 microM). During these low-level kainate exposures, large SMI-32(+) neurons showed higher intracellular Ca2+ concentrations than most spinal neurons, suggesting that Ca2+ ions are also important in this more slowly evolving injury.