Hemodialysis patients are at high risk of infection by hepatitis C virus (HCV). The aim of this study was to investigate an HCV outbreak that occurred in an autodialysis unit by using epidemiological and molecular methods. Seroconversion to HCV antibody (anti-HCV) was observed in two patients over an 18-month period; two other patients had previously been recorded as anti-HCV positive. All four patients involved in the outbreak were tested for HCV RNA, and hepatitis C genotype determination was accomplished by a reverse hybridization assay. Furthermore, part of hypervariable region 1 (HVR1) of the hepatitis C genome was amplified and sequenced in samples from all HCV RNA-positive patients. Phylogenetic analysis of the nucleotide sequences obtained was carried out in order to investigate any possible epidemiological linkages among patients. The nucleotide sequences of the HVR1 regions of both newly infected patients were found to be identical to sequences of samples from previously recorded anti-HCV-positive original patients, suggesting that they were infected by the same isolate. Molecular and epidemiological analysis suggested that nosocomial patient-to-patient transmission was the most likely explanation for the virus spread in the autodialysis unit under study.