To control Mycoplasma hyopneumoniae infections, the use of antibiotics in feed or water is a common practice. Unfortunately, information concerning the susceptibility of M. hyopneumoniae to different antimicrobials is very scarce. The in vitro susceptibility of 21 M hyopneumoniae field isolates was determined in this study using a broth microdilution technique. Acquired resistance to spectinomycin, oxytetracycline, doxycycline, gentamicin, florfenicol and tiamulin was not observed. One isolate showed acquired resistance to lincomycin, tilmicosin and tylosin, but was susceptible to all other antibiotics tested. For 5 isolates, the MIC-values of flumequine were > 16 mu g/ml, while the MIC50-value was 2 mu g/ml. The MIC-values of enrofloxacin for these 5 isolates were >= 0.5 mu g/ml, the MIC50 being 0.06 mu g/ml. This is the first report showing acquired resistance against macrolides, lincosamides and fluoroquinolones in M hyopneumoniae field isolates.