We analyzed the relationship between rifampin MICs and rpoB mutations of 40 clinical isolates of Mycobacterium tuberculosis. A point mutation in either codon 516, 526, or 531 was found in 13 strains requiring MICs of > or = 64 micrograms/ml, while 21 strains requiring MICs of < or = 1 microgram/ml showed no alteration in these codons. However, 3 of these 21 strains contained a point mutation in either codon 515 or 533. Of the other six strains requiring MICs between 2 and 32 micrograms/ml, three contained a point mutation in codon 516 or 526, while no alteration was detected in the other three. Our results indicate that the sequencing analysis of a 69-bp fragment in the rpoB gene is useful in predicting rifampin-resistant phenotypes.