Flavivirus proteins are produced by co- and post-translational proteolytic processing of a large polyprotein using both host- and virus-encoded enzymes. The flavivirus serine proteinase, which consists of NS2B and NS3, is responsible for cleavages of at least four dibasic sites in the nonstructural region. In this study, a number of substitutions for the conserved amino acids flanking the 3/4A and 4B/5 dibasic cleavage sites {Arg(P2)- Arg(P1)↓Gly(P1’)} were examined for their effects on yellow fever virus (YF) polyprotein processing. The substrate for these studies was a truncated YF polyprotein, called sig2A-5356, which consists of a signal sequence fused to NS2A and extending through the first 356 amino acids of NS5. At the P1’ position (Gly) of the 4B/5 site, only Ser and Ala were allowed while six other substitutions abolished cleavage. Substitutions of the 4B/5 P1 Arg residue with Lys, Gln, Asn, or His were tolerated while replacement with Glu eliminated cleavage. The 4B/5 P2 position (Arg) was found to be tolerant of substitutions with polar or hydrophobic residues which allowed varying degrees of partial cleavage. Previous studies have shown that cleavage at the 3/4A site is incomplete in YF-infected cells and that the cleavage efficiency at this site is significantly less for the sig2A-5356 polyprotein. Replacement of the 3/4A P1 Arg residue with noncharged polar or hydrophobic residues reduced the cleavage efficiency, whereas substitutions with Glu or Pro abolished cleavage. Studies with polyproteins containing one or both of the 3/4A and 4B/5 cleavage sites blocked indicate that there is not an obligatory processing order for cleavages generating the N termini of YF NS4A, NS4B, and NS5. © 1993 Academic Press, Inc.