Recently, we showed that yellow fever virus (YFV) E and NS1 proteins in Spodoptera frugiperda cells infected with a recombinant baculovirus are similar, if not identical to those produced during YFV infection. To study the role of E and NS1 in the induction of protective immunity against fatal YFV challenge, these viral antigens were expressed either alone or in tandem via recombinant baculoviruses Ac-E. NS1, Ac-E1 and Ac-NS1. Swiss mice were immunized with lysates of insect cells infected with the recombinant baculoviruses. Solid protection against lethal YFV encephalitis was achieved after immunization with cell lysates containing the E protein with or without the NS1 protein. Mice inoculated with recombinant protein NS1 alone were not significantly protected but showed an increased survival time. Recombinant E protein expressed alone or in tandem with NS1 elicited a low but significant level of neutralizing antibodies. Although protein NS1 synthesized by recombinant baculovirus expressing E plus NS1 was more immunogenic than that expressed alone, neither strategy induced NS1-specific antibodies with complement-mediated cytolytic activity.