The cause of amyotrophic lateral sclerosis (ALS) is still unknown. A possible relationship between ALS and sport participation has been supposed, but never definitely demonstrated. We studied a cohort of 7325 male professional football players engaged by a football team from the Italian First or Second Division in the period 1970–2001. ALS cases were identified using different concurrent sources. Standardized morbidity ratios (SMRs) were calculated. During the 137 078 person-years of follow-up, five ALS cases were identified (mean age of onset, 43.4 years). Three cases had a bulbar onset, significantly more than expected (P = 0.003). Since the number of expected cases was 0.77, the overall SMR was 6.5 [95% confidence interval (CI), 2.1–15.1]. The SMR was significantly increased for an ALS onset before 49 years, but not for older subjects. A significant increase of the SMR was found in the periods 1980–1989 and 1990–2001, whereas no ALS case was found in the 1970–1979 period. A dose–response relationship between the duration of professional football activity and the risk of ALS was found (>5 years, 15.2, 95% CI, 3.1–44.4; ≤5 years, 3.5, 95% CI, 0.4–12.7). Our findings seem to indicate that playing professional football is a strong risk factor for ALS.