Background: Nosocomial transmission of hepatitis C virus (HCV) in hemodialysis (HD) units is well established. In units with a high prevalence of HCV infection, the implementation of universal precautionary measures may not suffice in order to decrease the incidence and prevalence of HCV. In this setting strict isolation practices can be useful in order to achieve this goal. Methods: The incidence and prevalence of HCV infection amongst all HD and peritoneal dialysis (PD) patients from the province of Albacete, Spain, have been studied from 1992 to 2003.Through the 1993–1995 period chronic HD patients were treated either in a room exclusively for HCV– patients or in a room shared by HCV+ and HCV– patients. Complete separation of HCV+ and HCV– patients was implemented in 1995. Acute patients have been separated since 1992. The implementation of universal precautions was applied throughout the period. Results: There has not been a single seroconversion in the rooms where only HCV– patients were dialyzed during the 11 years of follow-up. There were two seroconversions in the rooms shared for 3 years by both HCV+ and HCV– patients. In 1995 the prevalence of HCV+ cases in HD and PD was 21.6 and 23.2%, respectively. Since then it has decreased steadily and in parallel for both therapies, and the current prevalence is 6.8% in HD and 5.7% in PD. Conclusions: In HD units with a high prevalence of HCV+ patients, strict isolation in combination with implementation of universal prevention measures can eliminate nosocomial transmission and obtain a long-term reduction in prevalence.