Extrahepatic replication may have important implications for the treatment of hepatitis C virus (HCV). Our aim was to analyse the association between the presence of positive/negative strand HCV RNA in different peripheral blood cell subsets at the end of PegIFN/RBV treatment, and treatment response in HIV-coinfected patients. Thirty-four HCV-HIV coinfected patients who concluded 48 weeks of PegIFN/RBV treatment were included in the present study. Positive/negative strand HCV RNA was detected by amplification of the 5' untranslated region (5' UTR) using high-temperature RT-PCR in immunomagnetic-isolated cell subsets. Twenty-three patients (67.6%) had sustained virologic response (SVR) while 11 patients (32.4%) relapsed. The frequency of positive/negative strand HCV RNA in any cell subsets was significantly lower in patients with SVR (8.6%) compared to relapsers (63.6%) (P = 0.002). Baseline HCV viral load was statistically higher among patients who relapsed (P = 0.008), while patients with SVR had very early virologic response more frequently (P = 0.003). Multivariate analysis showed, among these three variables, that only the presence of positive/negative strand HCV RNA was independently associated with relapse [P = 0.024; OR 14 (14-137)]. In conclusion, the presence of positive/negative strand HCV RNA at the end of treatment is associated with relapse among HCV-HIV coinfected patients and might have important implications in the clinical practice.