Processing of the nonstructural polyprotein of the hepatitis C virus (HCV) requires the serine-type proteinase located in the amino-terminal domain of NS3. To identify residues within NS3 determining substrate specificity, a mutation analysis was performed. Using sequence alignments and three-dimensional structure predictions, amino acids assumed to be important for specificity were replaced and the enzymes were tested in an intracellular trans-processing assay for their effects on cleavage of an NS4B-5B substrate. For some of the substitutions at positions 133, 134, 135, 136, 138, 152, 155, 157, and 169, slightly reduced processing efficiencies were observed but in no case was the substrate specificity altered. In contrast, substitutions of the phenylalanine at position 154 resulted in a modified cleavage pattern, suggesting an important role for this residue in substrate specificity. To substantiate this assumption, a panel of NS4B-5B substrates carrying different P1 residues at the NS4B/5A site were tested for cleavage by these altered proteinases. We found that substitution of Phe-154 by alanine, by valine, and particularly by threonine generated enzymes with the following affinities for aliphatic P1 residues: C > L > I > V for 154 F --> A, C = L > I > V for 154 F --> V and L > C > I > V for 154 F --> T. Neither leucine nor isoleucine nor valine was accepted by the parental NS3 proteinase, showing that Phe-154 is an important determinant for substrate specificity. Furthermore, we present evidence that Ala-157 plays an additional but minor role for this property.