A nationwide prospective survey on hepatitis C virus (HCV) infections among dialysis patients in The Netherlands was performed. Patients were recruited from 34 dialysis centers and were tested for antibodies and HCV RNA in 1995 and 1997. Seronegative serum samples were analyzed by reverse-transcriptase polymerase chain reaction in pools. HCV-RNA—positive serum samples were genotyped and were partly sequenced. In the first and second rounds, 67 (2.9%) of 2281 and 76 (3.4%) of 2286 patients were HCV positive, respectively. Of 960 patients with paired serum samples, 35 were HCV positive in both rounds, and 9 HCV-positive cases were newly identified in the second round. The incidence of HCV infection was 0.5 per 100 dialysis years. Phylogenetic analysis revealed clustered sequences that indicated nosocomial transmission. Sixty percent of HCV infections, however, can be attributed to 4 interdependent risk factors (i.e., hemodialysis before 1992, kidney transplantation before 1994, and birth or dialysis in a foreign country). In conclusion, the prevalence of HCV infections in The Netherlands does not decline, and transmission within dialysis units continues. Adequate screening of HCV infections and strict enforcement of universal infection control practices are required.