Mutations in the superoxide dismutase gene 1 (SOD-1) are found in patients with familial amyotrophic lateral sclerosis (FALS). Overexpression of a mutated human SOD-1 gene in mice results in neurodegenerative disease as result of motoneuron loss in lumbar spinal cord (10). Using this mouse model of FALS, we have established a quantitative assay utilizing the retrograde tracer Fluorogold (FG) to determine the number of motoneurons innervating one skeletal muscle in mice with ongoing disease. In adult wild-type mice, the number of α motoneurons retrogradely labeled by an injection of FG into medial gastrocnemius muscle is 50 ± 7 and this number remains constant from 7 to 18 weeks of age. In mutant mice, the number of α motoneurons retrogradely labeled by FG is the same as in wild-type mice at 7 and 9 weeks, but then declines to 36% of that in normal mice at 18 weeks. This decline also correlates positively to severity of motor impairments in these mice as assessed by the hindlimb splay test. In contrast, the number of FG-labeled γ motoneurons remains relatively unchanged in both wild-type and mutant mice up to 18 weeks. At 18 weeks of age, this apparent α motoneuron denervation is paralleled by an average of 55% reduction of MG-muscle mass and 40% weaker performance in the hindlimb splay test. These data suggest that α motoneurons are the most vulnerable neuronal subtype in this mouse model of ALS and it is primarily their loss that leads to functional motor deficits. This quantitative bioassay also will be valuable for evaluating novel therapeutics for ALS.