Monoclonal antibodies prepared against vaccine strains of yellow fever (YF) virus were initially characterized by fluorescence microscopy of Vero cells infected with YF virus strain 17D. When similarly tested against representatives of all flavivirus subgroups, the antibodies produced a wide spectrum of reactions ranging from the monospecific to the broadly cross-reactive; at least five antigenic domains in the YF virus envelope glycoprotein were identified. Monoclonal antibodies differentiated between YF virus vaccine strains (17D, 17DD, FNV), wild-type viruses and plaque variants selected from a 17D pool. One isolate from a patient with YF was antigenically similar to the Brazilian vaccine strain 17DD. Several of the antibodies reacting with the YF viral envelope glycoprotein in biological tests identified the 54K envelope glycoprotein; 45K and 26K polypeptides in YF 17D virus-infected cells were also identified by radioimmunoprecipitation and polyacrylamide gel electrophoresis. Neither of these polypeptides was found in uninfected cells. They may represent short-lived precursors of the 54K protein, post-translational cleavage or breakdown products. Other antibodies reacted with a 48K polypeptide in virus-infected cell lysates. This may be the non-structural NV3 protein described for YF virus. Its appearance on the surface of unfixed infected cells, but not on released virions, was demonstrated by fluorescence microscopy.