Necrotic enteritis, caused by netB toxin-producing Clostridium perfringens type A, is an important disease in broiler chickens worldwide. Earlier attempts to prevent necrotic enteritis by vaccination have not sufficiently taken into account the practical limitations of broiler vaccination. In most published studies on vaccination against necrotic enteritis, multiple doses at different ages are administered, which is not practical for broilers. The aim of this study was to compare the efficacy of subcutaneous single vaccination at day 1 or day 3 and double vaccination at day 3 and day 12, using crude supernatant containing active toxin or formaldehyde-inactivated supernatant (toxoid) of a netB-positive C. perfringens strain in a subclinical necrotic enteritis model. Double vaccination with crude supernatant resulted in a significant decrease in the number of chickens with necrotic enteritis lesions. The efficacy of vaccination using toxoid was lower compared with crude supernatant. Single vaccination with crude supernatant at day 3 resulted in significant protection, while vaccination of 1-day-old chickens with crude supernatant or toxoid, as envisaged for practical field application, did not induce protection.