The present study aimed to determine the prevalence and mechanisms of macrolide-lincosamide (ML) resistance in 72 Staphylococcus aureus isolates from cows with clinical mastitis. Minimum inhibitory concentrations (MIC) of ML antibiotics were determined by the broth microdilution technique, inducible ML resistance phenotype by the D test, and ML resistance genes by PCR assay. The isolates showed a high level of resistance to erythromycin (93.1%), azithromycin (93.1%), spiramycin (41.7%), tylosin (40.3%), tilmicosin (27.8%), and clindamycin (36.1%). Macrolide-lincosamide MIC(90) values were > or = 128 mg/L. Inducible ML resistance (iML) phenotype was detected in 52.8% (38/72) of isolates. In erythromycin-resistant (ER-R) strains, methylase genes ermB and ermC, efflux gene msrA/msrB, and inactivating enzyme genes lnuA and mphC were present alone or in various combinations, with ermB and ermC genes predominating. This is the first report of ML resistance genes ermB, mrsA/mrsB and mphC in S. aureus isolated from bovine mastitis. The occurrence of high levels of resistance to ML antibiotics among the S. aureus isolates, and the high rate of iML phenotype, indicate that appropriate alternative antibiotics should be prescribed for treating bovine mastitis caused by S. aureus. Furthermore, significant differences in the conformations of lactone rings of 16- and 14-membered macrolides could explain why some isolates with a constitutive ML resistance (cML) phenotype were sensitive to 16-membered macrolides alone. The different interaction of the 16-membered macrolides with the 50S ribosomal subunit is also presumably the reason why the susceptibility results of tilmcosin differed from those of tylosin and spiramycin.