Immunogenicity of an aqueous Mycoplasma gallisepticum (MG) bacterin and two oil-emulsified bacterins with aqueous-phase-to-oil-phase ratios of 1:2 and 1:4 were evaluated in 3 groups of 5-week-old MG-free White Leghorn chickens. Group 4 chickens were nonimmunized controls. Group 1, 2, and 3 chickens received primary immunizations with 0.5 ml of bacterin subcutaneously (SC). Six weeks later, half of the vaccinated chickens received a second immunization (0.5 ml SC). Six weeks after the last dose of vaccine, all 4 groups of chickens were challenged intraocularly with a 24-hr broth culture of pathogenic MG ("R' strain). The geometric mean hemagglutination-inhibition antibody titers (GMT) of non-immunized, once-immunized, and twice-immunized chickens were compared at 2-week intervals following primary immunization, secondary immunization, and challenge. Both oil-emulsified bacterins stimulated significantly higher (P less than or equal to 0.05) levels of antibody than the aqueous bacterin after the primary and secondary immunizations. Following challenge with pathogenic MG, the GMT in the immunized and non-immunized chickens approached comparable values. The highest GMT in groups 1, 2, 3, and 4 respectively were: a) primary response, 19, 99, 121, and 11; b) secondary response, 105, 423, 793, and 21; and c) challenge response, 171, 278, 300, and 160. After challenge there was a further rise of antibody level in non-immunized chickens, those immunized with the aqueous bacterin, and those immunized only once with the oil-emulsified bacterins. This suggested a lack of protection against challenge. However, there was no increase of GMT in chickens immunized twice with the oil-emulsified bacterins.