The highest prevalence of hepatitis C virus (HCV) infection among hemodialysis patients in Slovenia was found in a small dialysis unit with old equipment and room shortage, where the first anti-HCV-positive patient was detected in 1990. In 1992 and 1993, an additional 8 and 7 patients seroconverted, respectively. Genotyping analyses among 15 HCV RNA-positive patients showed quite unusual HCV genotype distribution for our country: genotypes 2 and 3 were determined in 9 and 6 patients, respectively. Sequence analysis of the 270-bp part of the NS-5 region was carried out in 12 patients. In 6 patients infected with subtype 2c and in 4 patients infected with subtype 3a, very similar sequences were obtained, forming two distinct clusters in the phylogenetic tree. In 2 patients infected with subtype 2c, viral strains were neither related to the main 2c strain nor to each other. Phylogenetic analysis unequivocally confirmed simultaneous nosocomial spread of two different HCV strains in one hemodialysis unit. The study confirmed that implementation of rigorous hygienic routines and introduction of separate rooms and machines for HCV-infected patients are important measures for effective control of HCV infection in a hemodialysis environment.