The P21 antigen of Toxoplasma gondii, defined by the monoclonal antibody TG17-113, has been described as a dense granule component, secreted in the parasitophorous vacuole during host cell invasion. The present work reports the cloning of the gene encoding the P21 antigen, for which we propose the name GRA 5. A cDNA library was screened with a rat antiserum raised against an HPLC fraction enriched in the P21 antigen. cDNA clones encoding GRA 5 were selected by antibody selection on the recombinant proteins. All these clones were incomplete at the 5' end. The 5' fragment of the longest cDNA clone isolated by this first screening was used as a probe in secondary screenings of cDNA and genomic DNA libraries. A genomic fragment containing the P21 gene and nearly full-length cDNAs have been isolated and sequenced. The gene encoding GRA 5 is 834 bp long and does not contain any intron. The deduced amino acid sequence of an open reading frame encoding 133 amino acids perfectly matched that of 5 peptides microsequenced from the native antigen. A N-terminal hydrophobic region was found to possess the characteristics of a signal peptide of 25 amino acids. A second hydrophobic domain, bordered by two hydrophilic regions strongly suggests a transmembrane region. This molecular structure is supported by ultrastructural studies showing the association of the P21 antigen with the parasitophorous vacuole membrane.