Groups of eight chickens were challenged with 10-fold dilutions of one of two strains of Mycoplasma synoviae (MS); each challenge group contained two noninfected sentinels. Both strains were highly efficient in colonizing the respiratory tract with challenge doses as low as 76 and 24 color-changing units/bird. Infection spread rapidly (within 7 days) to sentinels, while uninfected control chickens separated from infected chickens by two empty pens remained uninfected for the 56-day experimental period. Although sentinels and birds challenged with the lowest doses had weaker or slightly slower antibody responses in some cases as measured by serum plate agglutination, enzyme-linked immunosorbent assay (ELISA), and hemagglutination inhibition (HI), they generally exhibited a typical antibody response. Agglutination reactions tended to be weak, but a high percentage of tests (generally >30% from day 14 postchallenge) were positive. ELISA results were variable, and in some cases reactor rates were low (generally <20%), even though the chickens were colonized in the upper respiratory tract. The HI test was reliable in detecting infected groups; usually >50% were positive from 14 days postchallenge. Mean HI titers were higher when using hemagglutination antigens prepared from the homologous MS strain as compared with antigen prepared from the heterologous strain or with standard antigen prepared from WVU 1853.