In order to indicate the cross-resistance between rifampin (RMP) and rifabutin (RBU), minimal inhibitory concentrations (MICs) of RBU were investigated in 50 Mycobacterium tuberculosis strains. The MIC values of 25 RMP-susceptible (to 2 mug/ml) and 25 RMP-resistant (to 2 mug/ml) M. tuberculosis strains against RBU were determined by the Bactec TB 460 system. All of the RMP-susceptible strains were also susceptible to RBU (MIC less than or equal to1 mug/ml). Three out of 25 (12%) RMP-resistant strains were determined as susceptible to RBU. The high level cross-resistance (88%) obtained in this study highlights the importance of testing susceptibility to RBU prior to its inclusion in the tuberculosis treatment regimens at the Istanbul Faculty of Medicine.