Rifampicin resistance of Brucella melitensis by rpoB gene analysis has not yet been performed in Turkey, where brucellosis is endemic. In this study, we investigated the efficacy of E-test and single nucleotide polymorphism (SNP) analysis of the B. melitensis rpoB gene, for the detection of mutations conferring rifampicin resistance, by sequencing 21 human B. melitensis strains from the Southeast and Marmara regions of Turkey. On CLSI slow-growing bacteria standards, all isolates were sensitive to rifampicin except for 6 which showed intermediate resistance to rifampicin. MIC(50) and MIC(90) values were 1 mu g/ml and 1.5 mu g/ml respectively (range 0.50 -1.5 mu g/ml) The rifampicin-resistant phenotype was investigated at Cd 154 (GTT/(T) under bar TT), Cd 526 (GAC/(T) under bar AC, GAC/(A) under bar AC, GAC/G (G) under barC), Cd 536 (CAC/C (T) under barC, CAC/(T) under bar AC), Cd 539 (CGC/(A) under bar GC), Cd 541 (TCG/T (T) under barG) and Cd 574 (CCG/C (T) under barG) of the rpoB gene in B. melitensis 16M and B115 strains, and in clinical isolates. No missense mutations were found in any of the B. melitensis isolates, which indicates that all isolates were rifampicin-susceptible. In conclusion, SNP analysis was useful as a molecular tool for rifampin resistance testing. Although resistance to rifampicin was not detected in our strains of B. melitensis; the presence of strains with intermediate resistance to rifampicin indicates that susceptibility testing should be performed periodically.