The coding region of segment 4 of Thogoto (THO) virus, a tick-borne member of the Orthomyxoviridae, was expressed in a baculovirus system under the control of the polyhedrin promoter. This construct expressed authentic envelope glycoprotein as determined by size and antigenic reactivity with a panel of monoclonal antibodies (MAbs). Immunization of hamsters with Spodoptera frugiperda (Sf21) cells infected with the recombinant baculovirus induced neutralizing and protective antibodies against virus challenge; control hamsters developed clinical disease with high-titer viremia 3 days postchallenge. In contrast to hamsters, guinea pigs are comparatively resistant to THO virus infection but support nonviremic transmission between cofeeding infected and uninfected ticks. However, when uninfected ticks fed on guinea pigs immunized with the baculovirus recombinant, only 2% became infected following virus challenge of the animals compared to 26% of ticks on control nonimmune guinea pigs. Furthermore, neutralizing MAbs specific for THO viral glycoprotein protected mice against lethal challenge with THO virus; nonneutralizing MAbs specific for the glycoprotein, which inhibit THO viral agglutinating activity, did not induce a protective response. Thus at least in the murine model, protective immunity is conferred by antibodies directed against the neutralizing epitope(s) of the baculovirus-related glycoprotein of THO virus.