Real-time PCR was used to determine rifampin resistance in clinical isolates of Mycobacterium tuberculosis. Ninety-six rifampin-resistant isolates and 23 rifampin-susceptible isolates were included in the study. A 305-bp region covering the 81-bp "rifampin resistance-determining region" of rpoB was amplified. Two hybridization probe pairs that covered the most frequent mutation sites in rpoB, codon regions 526 to 531 and 513 to 516, were used. The results obtained by real-time PCR were compared to those obtained by the proportion method. For detection of rifampin resistance, the real-time PCR assay yielded a sensitivity of 92.7% and a specificity of 100%. Real-time PCR is a very rapid method, and it can be especially helpful for the reporting of resistant clinical isolates in a very short period of time.