Flavivirus proteins are produced by translation of a single long open reading frame and a complex series of cotranslational and post-translational proteolytic cleavages. To study these processing events in yellow fever virus (YF)-infected cells, polyclonal antisera recognizing C, prM, E, NS1, NS2B, NS3, NS4B, and NS5 were generated using peptide and fusion protein immunogens. Evidence suggests that production of the structural protein precursors involves rapid cotranslational processing consistent with signalase cleavages. The synthesis of the NS1 glycoprotein involves cleavage of polyprotein precursors (t1/2 approximately 10 minutes) which probably contain portions of the NS2A gene product. Endoglycosidase F treatment or labeling in the presence of tunicamycin suggests that YF prM and NS1 each have two N-linked oligosaccharides. NS2B is produced without any identifiable precursors or associated polyprotein species. Processing of the NS3-4-5 region is complex and occurs rapidly. A series of polyproteins can be detected whose molecular weights correlate with the cleavage sites defined by available N-terminal amino acid sequence data. However, convincing precursor-product relationships between these polyproteins and the mature NS3 and NS5 proteins could not be demonstrated. In contrast, NS4B appears to be produced by cleavage of a discrete precursor believed to be NS4AB. N-terminal sequence data for the putative NS4AB product has tentatively defined the NS3-4A cleavage site. A scheme for in vivo processing of the YF polyprotein is presented and discussed.