Identification of antigens that induce an immune response against tick infestations is required for the development of vaccines against these economically important ectoparasites. In order to identify protective antigens, we constructed a cDNA expression library from a continuous Ixodes scapularis cell line (IDE8) that was initially derived from tick embryos. cDNA clones were subjected to several rounds of screening in which mice were immunized with individual pools and then challenge-exposed by allowing I. scapularis larvae to feed on the immunized and control mice. Immunity against tick infestation was determined by the reduction in the ability of the larvae to feed to repletion and molt to the nymphal stage. Individual clones in pools that induced immunity to larval infestations were partially sequenced and grouped according to their putative protein function by comparison with sequence databases. The screening identified several individual antigens that induced a protective immune response against I. scapularis infestations. Our studies demonstrated for the first time that cDNA expression library immunization (ELI) combined with sequence analysis is a powerful and efficient tool for identification of candidate antigens for use in vaccines against ticks.