Mycoplasma synoviae (MS) strain MS-H was used in three separate commercial flocks for large-scale evaluation of the safety and efficacy of the vaccine under commercial conditions. MS-H successfully colonized meat and layer-breeders vaccinated by eyedrop and persisted for up to 55 wk after vaccination. Restriction fragment length polymorphism analysis showed that MS-H was the only strain isolated from two vaccinated flocks. In a third flock, challenge with a wild-type MS occurred, and this strain was isolated from both vaccinated and unvaccinated birds. Vertical transmission of MS-H was investigated by culturing pipped embryos and testing broiler progeny for MS antibody at processing (56 days old). No evidence of vertical transmission was detected. Lateral transmission of MS-H strain from vaccinated to unvaccinated birds occurred in one of the commercial flocks. Forty-one of 50 isolates of MS-H obtained from vaccinated flocks maintained their temperature-sensitive phenotype, but nine isolates showed a nontemperature-sensitive phenotype.