Mycoplasma bovis, a pathogenic mollicute first reported in 1961, is associated with pneumonia, mastitis, conjunctivitis, otitis, and arthritis in cattle. 8 Reports from Europe suggest that M. bovis is found in 13-50% of animals with disease. 13 It is unclear what percentage of disease in the United States may be attributed to M. bovis since large-scale epidemiological studies have not been performed. Poor efficacy of M. bovis vaccines produces few clinical options. Mycoplasma bovis infections are either treated with antimicrobials, which frequently do not clear the infection, or infected animals are culled. There is concern over the increasing resistance to frequently administered antimicrobials, including spectinomycin, tetracycline, and tilmicosin.14 The unique characteristics of M. bovis, such as the lack of a cell wall, cause concern regarding treatment options due to the ineffectiveness of cell wall-targeting antimicrobial therapies. Clinical Laboratory Standards Institute (CLSI)-approved and standardized levels of minimum inhibitory concentrations (MIC) for antimicrobials have not been established regarding Mycoplasma species.9,11,12 Multiple methods have been employed in order to determine MICs in many species of Mycoplasma. These methods have included broth dilutions, solid agar testing, E-strip testing, and most recently, flow cytometry. 2,6,9,10,15,16,18 The MIC values have been determined for Mycoplasma agalactiae, Mycoplasma hyopneumoniae, Mycoplasma putrefaciens, Mycoplasma capricolum, and Mycoplasma mycoides using a selection of antimicrobials with flow cytometry for one isolate of each. 1-3The objectives of the current study were to employ a microdilution method to determine the MIC of 7 antimicrobials on M. bovis isolates (n = 192) submitted to the Pennsylvania Animal Diagnostic Laboratory (PA-ADL; University Park, (n = 192) were tested for antimicrobial susceptibility to enrofloxacin, erythromycin, florfenicol, spectinomycin, ceftiofur, tetracycline, and oxytetracycline using a broth microdilution method. The most effective antimicrobials against M. bovis determined by using the broth microdilution method were florfenicol, enrofloxacin, and tetracycline with minimum inhibitory concentration (MIC) ranges of 2-32 µg/ml, 0.1-3.2 µg/ml, and 0.05 to >12.8 µg/ml, respectively. Spectinomycin, oxytetracycline, and tetracycline showed a wide-ranging level of efficacy in isolate inhibition with broth microdilution with MIC ranges of 4 to >256 µg/ml, 0.05 to >12.8 µg/ml, and 0.05 to >12.8 µg/ml, respectively. A significant difference in the susceptibility levels between quarter milk and lung isolates was found for spectinomycin. When MIC values of a subset of the M. bovis isolates (n = 12) were tested using a flow cytometric technique, the MIC ranges of enrofloxacin, spectinomycin, ceftiofur, erythromycin, tetracycline, oxytetracycline, and florfenicol ranges were 0.1-0.4 µg/ml, 4 to >256 µg/ml, >125 µg/ ml, >3.2 µg/ml, <0.025 to >6.4 µg/ml, 0.8 to >12.8 µg/ml, and <2-4 µg/ml, respectively. Flow cytometr...