The clinical correlation between the degree of HCV variability and the response to anti-HCV treatment in HIV positive patients infected with HCV genotype 3a is unknown. In this study, 27 HIV positive and 5 HIV negative patients with HCV genotype 3a infection were treated with interferon-alpha-2b with or without ribavirin. Nine patients (5 HIV positive) achieved a sustained virological response (SR) and 23 (only one HIV negative) were non-responders (NR). Sequence analyses of the partial E2 domain and the non-structural 5A protein were performed at baseline in all patients, and before and during treatment in the HIV positive NRs. There was no difference in the mean number of amino acid mutations from HCV 3a prototype, within E2 region, between the HIV positive and HIV negative patients: 17 (range 11-25) vs 16 (range 14-17). The mean baseline number of mutations in E2 region, was similar in HIV positive SRs and NRs: 18 (range 14-25) vs 16 (range 11-19). Phylogenetic analysis of HCV paired serum samples at baseline and during treatment revealed identical E2 sequence in 5/21 HIV positive NR patients, whereas 6 other sequences were strictly related to baseline E2 domain and the remaining 10 were divergent. The mean number of amino acid mutations in the NS5A protein at baseline, was 1 (range 0-3) in HIV negative patients and 2 (range 0-4) in HIV positive ones. This region was highly conserved in all isolates of HIV positive NRs analysed during treatment. These results suggest that genetic variability at baseline within the E2 region and NS5A protein of HCV 3a strain obtained from HIV positive and HIV negative patients is not associated with treatment response. Furthermore, the anti-HCV treatment did not influence HCV heterogeneity within the E2 and NS5A domains in HIV positive patients infected with HCV genotype 3a.