The NS3 serine protase of Hepatitis C virus (HCV) requires NS4A protein as a cofactor for efficient cleavage at four sites in the nonstructural region. The cofactor activity has been mapped to the central hydrophobic region (aa 22-34) of this 54-amino-acid NS4A protein, and site-directed mutagenesis has identified alternating hydrophobic amino acids, particularly Ile25 and Ile29, as critically important. A double mutant of NS4A cofactor peptide, I25A/I29A, completely abolished the cofactor activity. We now report that the cofactor peptide activity in the I25A/I29A double mutant can be restored specifically by introducing a biotin-aminohexanoic acid fusion at the N-terminus. In addition, a similar N-terminal fusion of biotin-aminohexanoic acid with the wild-type 4A peptide sig -nificantly enhanced cofactor activity. Our data corroborate the crystal structure-based hypothesis of hydrophobic interaction between the N-terminus of NS4A and the N-terminal alpha(0) helix of NS3 protease.