A series of fusion proteins corresponding to the hydrophobic ns2 and ns4 regions of yellow fever virus (YF) were generated in Escherichia coli using trpE fusion vectors. Antisera to ns2 and ns4 region fusion proteins recognize virus-specific proteins of 15 and 27 kDa, respectively. N-terminal amino acid sequence analysis of the 27-kDa protein indicates that the N-terminus of YF NS4B immediately follows a signalase-like cleavage site. Additional sequence data generated by microsequence analysis of labeled proteins immunoprecipitated with mouse hyperimmune antisera have identified the 15-kDa protein as NS2B and an additional 20-kDa viral protein as NS2A. Comparison of the sequences adjacent to the N-termini of these viral proteins suggests that three distinct types of cleavage events are involved in processing the hydrophobic YF ns2 and ns4 regions. These include cleavage after a short side chain amino acid to generate the N-terminus of NS2A, cleavage after two arginine residues to produce the N-terminus of NS2B, and a cleavage site consistent with the specificity of signalase to generate the N-terminus of NS4B. Analysis of virus-specific protein patterns in several different mammalian cell lines and in Aedes albopictus cells suggests that the same cleavage sites are used in different hosts. These findings are discussed in relation to the processing of flavivirus polyproteins.