We previously showed that two proteins, a 56-kDa protein (p56) and a 58-kDa protein (p58), are produced from the hepatitis C virus (HCV) nonstructural 5A region (NS5A) and that the production of p58 is enhanced by the presence of NS4A (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320-326, 1994). Both proteins have phosphorylated serine residues, some of which are located in the C-terminal region. In p58, phosphorylation of serine residues in the central region of HCV NS5A is important for production of p58 in an NS4A-dependent manner. To clarify the mechanism of NS5A phosphorylation, in particular phosphorylation in the central region, phosphorylation of deleted and mutated forms of NS5A was analyzed using a transient protein production system in cultured cells in the presence or absence of NS4A. Association of the NS5A region from amino acids 2135 to 2139 with NS4A was important for NS4A-dependent phosphorylation of NS5A.