The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly ␣-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1 H and 13 C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal ␣-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism. Hepatitis C virus (HCV)8 infection is a major cause of human chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1). About 170 million individuals worldwide are chronically infected with HCV, and current therapy based on a combination of pegylated interferon and ribavirin is poorly tolerated and ineffective in 50% of patients. In this context, the ongoing search for new drugs and targets is very active, and the structural and functional characterization of the HCV viroporin p7 is essential for the molecular understanding of its role in HCV replication and for antiviral drug development.HCV is a highly variable enveloped positive-stranded RNA virus, and patient isolates are classified into seven genotypes and numerous subtypes (2, 3) within the genus Hepacivirus of the family Flaviviridae (4). The HCV genome encodes a polyprotein precursor,