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Background & Aims Hepatitis C virus (HCV) poses a global health problem, with over 170million chronically infected individuals at risk of developing progressive liver disease. The ability of a virus to spread within a host is a key determinant of its persistence and virulence. HCV can transmit in vitro by cell-free particle diffusion or via contact(s) between infected and naive hepatocytes. However, limited information is available on the relative efficiency of these routes, our aim is to develop physiologically relevant assays to quantify these processes. Methods We developed a single-cycle infection assay to measure HCV transmission rates. Results We compared HCV spread in proliferating and arrested cell systems and demonstrated a significant reduction in cell-to-cell infection of arrested target cells. Comparison of cell-free and cell-to-cell virus spread demonstrated relatively poor transmission rates, with 10–50 infected producer cells required to infect a single naive target cell. We found HCV strain J6/JFH to be 10-fold more efficient at spreading via the cell-to-cell route than cell-free, whereas SA13/JFH and HK6/JFH strains showed comparable rates of infection via both routes. Importantly, the level of infectious virus released from cells did not predict the ability of a virus to spread in vitro , highlighting the importance of studying cell-associated viruses. Conclusions These studies demonstrate the relatively poor infectivity of HCV and highlight differences between strains in their efficiency and preferred route of transmission that may inform future therapeutic strategies that target virus entry.