The detection of hepatitis C virus (HCV) RNA by nucleic acid amplification techniques is the method of choice to differentiate between ongoing and past infection, and can be used to monitor the course of HCV infection. In this study, we evaluated the performance characteristics of a newly developed transcription-mediated amplification (TMA)-based assay, the VERSANT HCV RNA qualitative assay, which was designed to qualitatively detect HCV RNA. Samples tested by the TMA assay included 100 HCV antibody negative sera; serial dilutions of an HCV genotype 1a panel; the WHO HCV RNA standard 76/790; an HCV genotyping panel; and 150 clinical specimens, including sera from patients who had received alpha interferon (IFN) treatment or liver transplants. TMA test results were compared with the Cobas Amplicor HCV polymerase chain reaction (PCR) assay. The analytical specificity of the HCV TMA assay was > 98%. No carry-over contaminations were observed. The assay demonstrated an analytical sensitivity of 100% at 41 HCV RNA copies/mL (genotype 1a panel) and 5 IU/mL (WHO standard), respectively. HCV genotypes and subtypes did not affect the results. Qualitative RNA detection by diagnostic Amplicor PCR and TMA was in agreement in > 97% of all 150 clinical samples tested. In our study, the TMA-based assay proved to be a specific and sensitive method for qualitative HCV RNA detection. The test may turn out to be an attractive alternative to already established techniques for HCV RNA amplification in routine clinical laboratories.