This study was conducted to compare the Minimal Inhibitory Concentrations (MICs) for enrofloxacin, lincomycin, tetracycline, tiamulin and tylosin, of Mycoplasma hyosynoviae, isolated from pigs at notably different intervals (1968-71 and 1995-96). Each group comprised 21 low passage isolates and a Danish reference strain (M60) and the type strain (S16). MICs were determined in liquid medium with both initial and final readings. Enrofloxacin, lincomycin, tetracycline and tiamulin were active against all isolates, and tiamulin showed the highest activity. For tylosin all the isolates from 1968-71 were highly susceptible, whereas the isolates from 1995-96 could be divided into a highly susceptible (nine isolates) and relatively resistant (12 isolates) group. This difference between old and new strains was statistically significant (p = 0.0000415). The remaining agents, enrofloxacin, lincomycin, tiamulin and tetracycline, showed an unaltered good activity against M. hyosynoviae. The resistance to tylosin seems now to occur so often that this antibiotic cannot be recommended for therapeutic use any more. The most probable explanation for the emergence of resistance is the intensive use of tylosin during many years for therapy and growth promotion.