We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.The year 2002 saw the publication of the genomic sequences of the human malaria parasite Plasmodium falciparum (12) and the rodent parasite Plasmodium yoelii yoelii (5). The hope is that this information will bring insights into parasite biology and lead to the development of new vaccines and drugs. However, novel research approaches are required to efficiently study the thousands of genes. This paper describes the development of a high-throughput technique for the identification of vaccine target antigens among newly annotated malaria genes. Our method rapidly produces large numbers of DNA vaccines carrying P. y. yoelii exons and measures their ability to reduce parasite load in mice. We call this screening technique the antigen identification method.The novelty and efficiency of the antigen identification method come from a combination of rapid production of DNA vaccines and sensitive measurement of parasite killing. With the annotated P. y. yoelii genomic sequence, we identify P. y. yoelii genes expressed during the sporozoite stage by comparison with expressed sequence tags (ESTs) generated from a cDNA library of P. yoelii sporozoites (20). PCR primers for these sporozoite P. y. yoelii genes are synthesized to be compatible with the Gateway cloning system, which allows rapid production of DNA vaccine plasmids. Mice are immunized with the DNA...