Summary Gel filtration through Sephadex G-100 was used to achieve a high degree of separation of the neurotoxin from the hemolysin of a crude concentrated culture filtrate of C. tetani. Recovery of the neurotoxin was complete but only 20% of the hemolysin was recovered. The fraction with maximum neurotoxic activity was essentially free of hemolysin while a thousand-fold increase in ratio of hemolytic units to neurotoxin was detected in the fraction with maximum hemolytic activity.