The Clostridium perfringens pore-forming toxin, NetB, is a major virulence factor in the pathogenesis of necrotic enteritis in chickens and hence is an interesting protein to evaluate as a vaccine antigen. In vitro grown cultures of C. perfringens produce very little NetB and hence do not provide a good source of the protein for use in vaccines. Therefore, we used recombinant versions of the mature NetB protein to investigate its efficacy in a variety of different vaccine formulations. Birds vaccinated with recombinant NetB (rNetB) were protected from a mild necrotic enteritis challenge, but this single subunit vaccine did not offer protection when a strong C. perfringens challenge was applied. However, statistically significant levels of protection from a strong heterologous challenge were achieved when the vaccine formulation was enhanced by combining rNetB with whole cell proteins (bacterin) or concentrated and inactivated C. perfringens culture supernatant (toxoid). Clostridial vaccines have been effectively used in other animal species for many years, but the traditional toxoid style of vaccine does not provide very effective protection against necrotic enteritis in chickens. We now have shown that supplementation of such vaccines with sufficient quantity of a key antigen (NetB) provides good levels of protection. We have gone on to show that these vaccines produce high antibody titres in hens and in their eggs and hatched chicks. In conclusion, the identification of a key virulence factor in the pathogenesis of necrotic enteritis (NetB) has led to the development of an effective vaccine formulation that will be of major value to the poultry industry.