Macrolides and related antibiotics are used to control mycoplasma infections in the pig industry worldwide. Some porcine mycoplasmas, however, survive these treatments by acquiring resistance. The mechanism of acquired resistance to macrolides and lincosamides was studied in more detail for Mycoplasma hyopneumoniae by comparing both the phenotype and genotype of a resistant field isolate to five susceptible isolates. The MICs were significantly higher for the resistant strain for all antibiotics tested. The MICs for the 16-membered macrolide tylosin ranged from 8 to 16 microg for the resistant strain and from 0.03 to 0.125 microg/ml for the five susceptible strains. The MICs for the 15-membered macrolides and lincosamides were higher than 64 microg/ml for the resistant strain while only 0.06 to 0.5 microg/ml for the susceptible strains. Mycoplasma hyopneumoniae strains are intrinsically resistant to the 14-membered macrolides due to a G 2057 A transition (E. coli numbering) in their 23S rDNA. Therefore, high MICs were observed for all strains, although the MICs for the resistant strain were clearly increased. An additional, acquired A 2058 G point mutation was found in the 23S rRNA gene of the resistant strain. No differences linked to resistance were found in the ribosomal proteins L4 and L22. The present study showed that 23S rRNA mutations resulting in resistance to macrolides and lincosamides as described in other Mycoplasma spp. also occur under field conditions in M. hyopneumoniae.