A role for the large glycoprotein (G1) of California encephalitis (CE) virus was examined in the infection of baby hamster kidney (BHK-21) and Aedes albopictus (C6/36) cell lines and the mosquito Ae. dorsalis using G1 monoclonal antibodies (MAbs) and selective protein cleavage. Five MAbs neutralized CE viral infectivity in both cell lines. One MAb, 7D4.5, efficiently neutralized the peroral infection of Ae. dorsalis females fed CE virus in artificial bloodmeals. To determine if MAbs to G1 neutralized CE virus by sterically hindering the small glycoprotein (G2), portions of G1 were trypsinized, and viral infectivity was assayed in vivo and in vitro. Cleavage of G1 resulted in a complete loss of infectivity both in mosquitoes and in culture, even though a significant amount of G2 remained intact. The loss of infectivity by both neutralization with G1 MAbs and trypsinization indicates that the G1 protein of CE virus is required for infection of mosquito and mammalian cells in vitro and of mosquitoes by the peroral route.