Lyme disease is sometimes part of the differential diagnosis for amyotrophic lateral sclerosis (ALS). Herein we report on 414 individuals with ALS at the Massachusetts General Hospital who underwent laboratory testing for Lyme disease. Twenty-four (5.8%) were seropositive, but only 4 (0.97%) had confirmed past immunoreactive infection. Two of these patients received ceftriaxone for 1 month without clinical improvement. Lyme disease was rare in 414 patients with ALS and is not likely to be causative.