The Siemens VERSANT® transcription‐mediated amplification (TMA) assay is extremely sensitive for the detection of hepatitis C virus (HCV) RNA in serum. Eleven of 180 subjects in the Hepatitis C Antiviral Long‐term Treatment against Cirrhosis (HALT‐C) Trial who achieved polymerase chain reaction (PCR)‐defined sustained virological response (SVR) at week 72 also had TMA‐positive results from the same blood draw; six were positive on repeat testing. We report the follow‐up on these 11 patients, and the reproducibility of TMA test results from PCR‐negative samples in relationship to antiviral treatment outcome. Peginterferon and ribavirin treatment was initiated in 1145 prior interferon nonresponders with advanced hepatic fibrosis. Treatment was continued for 48 weeks if patients had undetectable HCV RNA by PCR at treatment week 20. Frozen serum samples from weeks 12, 20, 24, 48, and 72 were subsequently tested by TMA. Nine of the 11 patients returned for testing (median, 30 months after the week 72 visit), and all had undetectable HCV RNA by TMA and PCR. Among 759 PCR‐negative samples obtained during treatment that were tested twice by TMA, 17% overall exhibited consistently positive results, and 21% exhibited inconsistently positive results. SVR was more likely if TMA was consistently negative than if consistently or inconsistently positive. With continued treatment, patients with inconsistently positive TMA results were more likely to become TMA‐negative than TMA‐positive (P < 0.0001). Conclusion: In PCR‐negative samples, positive TMA results may indicate the presence of low levels of HCV RNA. However, because patients with positive TMA results may achieve SVR, management decisions during therapy should not be based on a single positive TMA test result. (HEPATOLOGY 2008.)