To assess whether the occurrence of rifampicin (RFP) resistance in methicillin-resistant Staphylococcus aureus (MRSA) is related to treatment of tuberculosis, we determined the RFP susceptibility of MRSA isolates obtained from tuberculosis patients and screened for mutation(s) in the rpoB gene of these isolates. The MICs of RFP for 84 MRSA isolates obtained from two hospitals in Japan were determined. DNA was sequenced in the region 1318-1602 nucleotides (nt) of the rpoB gene, which includes RFP resistance-determining clusters I (1384-1464 nt, 462-488 amino acids). The majority of MRSA isolates from tuberculosis wards, i.e., 48 of 51 (94%) [33 of 34 in a Tokyo hospital (97%) and 15 of 17 in a Chubu hospital (88%)], were resistant to RFP. Meanwhile, no isolates of 33 from the other wards were resistant to RFP. All RFP-resistant MRSA isolates had a mutation(s), including novel mutation(s) such as Val453-->AEPhe, Asp471-->AEAsn, and Ile527-->AELeu, in rpoB. An emergence of RFP-resistant MRSAs in tuberculosis wards in Japan was strongly suggested.