Background: Methicillin-resistant S. aureus (MRSA) has been endemic in Hospital Universitari de Bellvitge, Barcelona, since 1990. During the 1990-95 period the Iberian clone (ST-247; SCCmec-I) was dominant. Isolates of clonal complex 5 (ST-125; SCCmec-IV) gradually replaced the Iberian clone from 1996 to 2003. A new multiresistant MRSA phenotype showing rifampicin resistance emerged in 2004 and rapidly increased from 25% in 2004 to 45% in 2006. The aims of this study were i) the molecular characterisation of rifampicin resistant MRSA isolates, ii) the study of the rifampicin resistance expression by disk diffusion, microdilution and E-test, and iii) the analysis of the rpoB gene mutations involved in rifampicin resistance. Results: A sample of representative 108 rifampicin-resistant MRSA isolates belonged to a single PFGE genotype, ST-228, SCCmec type I and spa type t041. Of 108 isolates, 104 (96%) had a low-level rifampicin resistance (MICs, 2 to 4 mg/L) and 4 a high-level rifampicin resistance (MICs, 128 - ≥ 256 mg/L). Disk diffusion and E-test methods failed to identify a low-level rifampicin resistance in 20 and 12 isolates, respectively. A low-level rifampicin resistance was associated with amino acid substitution 481His/Asn in the beta-subunit of RNA polymerase. Isolates with a high-level rifampicin resistance carried additional mutations in the rpoB gene. Conclusions: The emergence of MRSA clone ST228-SCCmec I, related to the Southern Germany clone, involved a therapeutical challenge for treating serious MRSA infections. Decreased susceptibility to rifampicin in MRSA strains of ST228-SCCmecI was associated with one or two specific mutations in the rpoB gene. One fifth of isolates with low-level rifampicin-resistance were missed by the diffusion methods.