Three chimpanzees persistently infected with hepatitis C virus of genotype II/1b were challenged with hepatitis C virus of genotype III/2a and 6 wk later with hepatitis C virus of genotype I/1a. They were tested for titers of total and genotype-specific hepatitis C virus RNA, as well as for serum transaminase levels, until 52 wk after the first challenge. One chimpanzee (CH489) with intermittent low hepatitis C virus RNA titers of genotype II/1b in serum was superinfected with hepatitis C virus of genotype III/2a between wk 1 and 7 after the challenge; superinfection was accompanied by fluctuating high transaminase levels. Later, the animal was superinfected with hepatitis C virus of genotype I/1a. Superinfection was accompanied by persistently high transaminase levels immediately after the challenge. Hepatitis C virus of genotype I/1a persisted, whereas hepatitis C virus of genotype II/1b was undetectable 22 wk after the challenge and thereafter. In another chimpanzee (CH353) with intermittent low hepatitis C virus RNA titers of genotype II/1b, hepatitis C virus of genotype III/2a induced fluctuating high levels of serum transaminases without revealing itself in serum. Then, HCV of genotype I/1a superinfected her, induced persistently high transaminase levels and took over HCV of genotype II/1b at 22 wk after the challenge and thereafter. The third chimpanzee (CH451) with persistently high HCV RNA titers of genotype II/1b did not reveal HCV RNA of genotype III/2a in serum after the challenge, although transaminases sharply increased. Low-titered HCV RNA of genotype I/1a was detected at 18 wk after the challenge. He continued to have elevated transaminase levels and high-titered HCV RNA of genotype II/1b throughout the observation period. These results indicate superinfection with HCV of different genotypes in chimpanzees, newly introduced HCV replacing or being expelled by the predecessor HCV with each infection inducing an episode of liver injury. (Hepatology 1994;20:1131–1136).