A commercial assay (Inno-Line Probe Assay; Innogenetics, Belgium) was evaluated to determine its ability to detect rifampin resistance in Mycobacterium tuberculosis directly from clinical specimens. Fifty-nine selected specimens (42 respiratory and 17 nonrespiratory) culture positive for Mycobacterium tuberculosis were tested along with their corresponding isolates in culture. The results were compared with those obtained by in vitro susceptibility testing. The results of the line probe assay to detect rifampin resistance in Mycobacterium tuberculosis present in clinical specimens and in cultured isolates were concordant for 58 of 59 (98.3%) isolates (95% confidence limits = 90.9-99.9%). The line probe assay failed only once, when a fecal specimen was tested; no amplification was observed due to the presence of inhibitory compounds. The most frequently observed mutation was His526-->Asp (58.7%), followed by the His526-->Tyr (23.9%); together, they represented 82.6% of rifampin-resistant samples. In conclusion, the Inno-Line Probe Assay is a rapid, useful method for detecting the presence of Mycobacterium tuberculosis complex and its resistance to rifampin directly from clinical specimens and culture. Moreover, since rifampin resistance is a potential marker for multidrug resistance in Mycobacterium tuberculosis, this assay may constitute an important tool for the control of tuberculosis.