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Background: Chronic hepatitis C virus (HCV) infection has occasionally been associated with diseases of the central nervous system, thus suggesting that the virus has a direct effect on cerebral function., Objective: To investigate the presence and heterogeneity of HCV populations in the cerebrospinal fluid (CSF), plasma and peripheral blood mononuclear cells (PBMC) of HIV-infected and anti-HCV-positive patients., Methods: Reverse transcriptase-polymerase chain reaction was used to detect HCV RNA in 21 paired CSF/plasma samples and 20 PBMC samples from HIV/HCV-positive patients. HCV was genotyped by means of a hybridization assay, and its compartmental heterogeneity was analysed by cloning the related amplicons., Results: All of the plasma samples, 14 out of 20 PBMC samples and five out of 21 CSF samples were positive for HCV RNA. Of the five patients with HCV-positive CSF, two had the same genotype in the plasma/CSF/PBMC samples, two had a different genotype in the CSF from that in plasma and PBMC, and one had the same genotype in paired CSF/plasma samples. An analysis of the HCV populations showed that two patients seemingly infected with the same genotype in different compartments harbored mixed infection in the CSF but not in the plasma and PBMC samples., Conclusions: These findings of different HCV genetic diversification in different compartments suggest that CSF is an independent site of viral replication and persistence.