Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis, immobilize the parasite in vitro. In order to identify cell surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-114 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1- and 2-dimensional SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.