Six major hepatitis C virus (HCV) genotypes and numerous subtypes have been described, and recently a seventh major genotype was discovered. Genotypes show significant molecular and clinical differences, such as differential response to combination therapy with interferon‐α and ribavirin. Recently, HCV research has been accelerated by cell culture systems based on the unique growth capacity of strain JFH1 (genotype 2a). By development of JFH1‐based intergenotypic recombinants containing Core, envelope protein 1 and 2 (E1, E2), p7, and nonstructural protein 2 (NS2) of genotype 6a and 7a strains, as well as subtype 1b and 2b strains, we have completed a panel of culture systems for all major HCV genotypes. Efficient growth in Huh7.5 cells depended on adaptive mutations for HK6a/JFH1 (6a/2a, in E1 and E2) and J4/JFH1 (1b/2a, in NS2 and NS3); viability of J8/JFH1 (2b/2a) and QC69/JFH1 (7a/2a) did not require adaptation. To facilitate comparative studies, we generated virus stocks of genotype 1–7 recombinants with infectivity titers of 10^3.7 to 10^5.2 50% tissue culture infectious dose/mL and HCV RNA titers of 10^7.0 to 10^7.9 IU/mL. Huh7.5 cultures infected with genotype 1–6 viruses had similar spread kinetics, intracellular Core, NS5A, and lipid amounts, and colocalization of Core and NS5A with lipids. Treatment with interferon‐α2b but not ribavirin or amantadine showed a significant antiviral effect. Infection with all genotypes could be blocked by specific antibodies against the putative coreceptors CD81 and scavenger receptor class B type I in a dose‐dependent manner. Finally, neutralizing antibodies in selected chronic phase HCV sera had differential effects against genotype 1–7 viruses. Conclusion: We completed and characterized a panel of JFH1‐based cell culture systems of all seven major HCV genotypes and important subtypes and used these viruses in comparative studies of antivirals, HCV receptor interaction, and neutralizing antibodies. (HEPATOLOGY 2009.)