Like most positive-strand RNA viruses, hepatitis C virus (HCV) forms a membrane-associated replication complex consisting of replicating RNA, viral and host proteins anchored to altered cell membranes. We used a combination of qualitative and quantitative electron microscopy (EM), immuno-EM, and the 3D reconstruction of serial EM sections to analyze the host cell membrane alterations induced by HCV. Three different types of membrane alteration were observed: vesicles in clusters (ViCs), contiguous vesicles (CVs), and double-membrane vesicles (DMVs). The main ultrastructural change observed early in infection was the formation of a network of CVs surrounding the lipid droplets. Later stages in the infectious cycle were characterized by a large increase in the number of DMVs, which may be derived from the CVs. These DMVs are thought to constitute the membranous structures harboring the viral replication complexes in which viral replication is firmly and permanently established and to protect the virus against double-stranded RNA-triggered host antiviral responses.