A systematic virological follow-up of hemodialysis patients identified 11 cases of de novo hepatitis C virus (HCV) infection in the same unit that were not due to blood transfusion. There were three groups of infection, each occurring within a period of 3 months: four infections with genotype 1b, two infections with genotype 1b, and five infections, four with genotype 1a and one with genotype 5a. The possibility of patient-to-patient transmission was addressed by sequencing the first hypervariable region of the HCV genome in sera taken shortly after infection. Phylogenetic analysis indicated clustering of most of the cases of de novo infections. Sequence homologies identified potential contaminators among already infected patients. All patients who were infected with closely related HCV isolates were found to have been treated in the same area and during the same shift or on the previous one. These infections could have been due to occasional breaches of the usual hygiene measures. Strict adhesion to hygiene standards and routines, continuously supervised, remains the key rule in the management of dialysis patients. Nevertheless, the isolation of patients with HCV could reduce the risk of infection because occasional lapses of preventive hygiene measures or unpredictable accidents can always take place in a hemodialysis unit. This policy needs to be evaluated by large-scale prospective studies.