The duration of protective immunity elicited by the MS-H vaccine was evaluated by experimental challenge of chickens at 15 and 40 wk after eyedrop vaccination. Immunity induced by the parent strain of the vaccine, 86079/7NS, was also investigated for comparison. A serological response to Mycoplasma synoviae was detected in 89% to 100% of MS-H vaccinates and 86079/7NS inoculates at 15, 27, 30, 35, and 40 wk after inoculation. A significantly lower incidence of air-sac lesions and lower air-sac lesion severity were observed in both the MS-H vaccinated and the 86079/7NS inoculated groups, as compared to the unvaccinated controls, after both challenge points. Tracheal mucosal thicknesses in MS-H vaccinates was significantly lower in the upper, lower, and total trachea at 40 wk after vaccination, as compared to the controls. It was demonstrated in this experiment that protective immunity, as determined by protection against experimental challenge, was maintained to at least 40 wk after vaccination.