Recent evidence of a protective immune response to the flavivirus non-structural protein, NS1, has suggested its incorporation into possible recombinant vaccines. The region of the 17D yellow fever virus (YFV) genome encoding the C terminus of envelope glycoprotein and extending to the N terminus of non-structural protein NS3 (NS1-NS2a-NS2b; nucleotides 2030 to 4940) was expressed in vaccinia virus and physical and immunogenic properties of the NS1 moiety were studied. Recombinant NS1 protein, and native YFV NS1, was detected at the surface of infected cells by immunofluorescence and by immune cytolysis after treatment with anti-NS1 antibody and complement. NS1 was also detected in the extracellular medium as a secreted form. Recombinant NS1 was immunoprecipitated as a single protein of approximately the same size as native 17D YFV NS1. Unboiled, both recombinant and native NS1 formed polymers of high Mr. Immunization of mice with this recombinant vaccinia virus stimulated production of non-neutralizing, complement-fixing cytolytic antibody and conferred partial protection against lethal intracerebral inoculation of mice with live 17D YFV.