Amyotrophic lateral sclerosis (ALS) is characterized by increased excitability of motor neurons and early involvement of large motor fibers that have low electrical thresholds. Despite the advent of new techniques of threshold tracking, exploration of this abnormal excitability has not been straightforward, by tracking at the single target level as previous reported, because of the heterogeneous nature of the disease process among fibers that have variable thresholds. We have assessed different populations of motor axons by tracking at four different target response levels (10, 20, 40 and 60% of maximum compound muscle action potentials), and conducted multiple nerve excitability tests in 27 ALS patients and 23 control subjects. In normal controls, axons with low thresholds have the following characteristics compared to those with high thresholds: greater threshold reduction during depolarizing currents and smaller threshold increase to hyperpolarizing currents, reflecting the order of the fiber size. In contrast, ALS patients lacked these relationships, suggesting increased variability of axonal membrane potentials. Three ALS patients demonstrated changes in threshold electrotonus, consistent with overt membrane depolarization, as seen in ischemic nerves. The variability of motor nerve excitability accounts for fasciculations, confirms previously reported dysfunction of potassium channels, and suggests failure of Na(+)/K(+)pumps, possibly caused by mitochondrial dysfunctions at the early stage.