To investigate whether distinct compositions of viral quasispecies developed in the ascitic fluid of patients with late-stage chronic hepatitis C virus (HCV) infection, samples of various origins, including ascitic fluid and ascitic mononuclear cells (AMCs), were analyzed by the method of single-strand conformation polymorphism (SSCP). Subsequently, the major species were isolated, sequenced, and subjected to phylogenetic analysis. Of 42 patients analyzed, HCV-RNA was detectable in the AMCs of 25 patients. SSCP analysis indicated that the compositions of viral quasispecies among samples of different origins were markedly different in this group of patients. Phylogenetic analysis revealed that the ascitic-fluid-derived clones were most closely related to the AMC-derived clones. Minus-strand HCV-RNA was detectable in 5 of them. Our data suggest that HCV can replicate in the AMCs of patients with late-stage chronic HCV, which results in the development of distinct viral quasispecies.