Mycoplasma synoviae (MS) was isolated from a flock of commercial tom turkeys in which a small percentage of the birds exhibited clinical signs and lesions typical of MS synovitis. However, serologic testing of such flocks revealed poor to inconsistent reactivity by agglutination, enzyme-linked immunosorbent assay (ELISA) or hemagglutination inhibition; isolation of MS from such flocks proved to be very difficult. Turkeys were challenged with one of the isolates (K4463B) either by aerosol or systemically by a combination of intravenous, foot pad, and eyedrop routes. Turkeys challenged by the systemic route responded normally to all serologic tests, whereas those challenged by aerosol either responded very poorly on all serologic tests or were seronegative up to 6 wk postchallenge even though they were positive for MS by tracheal culture. These results suggest that turkeys may harbor an upper respiratory infection with MS while remaining serologically negative.