Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation. By selecting for replication-competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular-dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19 aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity.