Toxoplasma gondii is a ubiquitous unicellular eukaryotic parasite with a complex intracellular life cycle, capable of invading and chronically infecting a wide variety of vertebrate host species including man. While normally opportunistic in healthy adults, it is a lethal pathogen in immunocompromised humans, particularly in AIDS-patients. We present the application of genomic phage display as a tool for the direct identification of antigens with potential value in diagnosis and/or as subunit vaccine component. Using a polycosmid cloning strategy, we constructed a large phagemid display library (> 10 exp 9 independent clones) of mixed short genomic restriction fragments (=< 500 bp) of T. gondii genomic DNA (80 Mbp genome size) fused to gene III of the filamentous phage M13. Biopanning of the library with monoclonal Toxoplasma antibodies resulted in the isolation and identification of an epitope of GRA3, an antigen located in the dense granules of T. gondii tachyzoites. The reactivity of the phage displaying the GRA3 epitope with the monoclonal antibody was confirmed by ELISA. These results demonstrate the accessibility of mid-sized eukaryotic genomes to display technology and the feasability to screen these whole genome display libraries with antibodies for isolating novel antigenic determinants.