Hepatitis C virus (HCV) encodes a polyprotein that is processed to produce the structural and nonstructural proteins of the virus. Nonstructural protein 3 (NS3) is a serine proteinase that cleaves the polyprotein to release the NS4A, NS4B, NS5A, and NS5B proteins. To characterize the substrate specificity of NS3, we synthesized by in vitro translation the polyprotein NS2*-NS3-NS4*P that includes 70% of the NS2 protein, the complete NS3 protein, and 25% of the NS4 protein region attached to substance P, an epitope tag. We demonstrated that NS3 cleaves at the NS3/NS4A junction to release the NS4*P protein. Subsequently, we used this reaction to evaluate the importance of conserved amino acids that flank the NS3/NS4A junction. We replaced amino acids in the P6, P1, and P1' positions of the scissile bond of this junction using site-directed mutagenesis. When the P6 aspartic acid was changed to asparagine, lysine, or serine, NS3-mediated cleavage occurred. When threonine in the P1 position was replaced with other polar amino acids or with amino acids having aliphatic side chains, cleavage occurred, although it was not detected when arginine or tyrosine was present. Replacement of serine in the P1' position with other polar amino acids, with amino acids having aliphatic side chains, or with arginine resulted in NS3-mediated cleavage. Thus, since fewer amino acids in the P1 position supported cleavage than in the P6 or P1' positions, the P1 position of the scissile bond may play a more important role in defining the substrate specificity of the HCV NS3 proteinase.