We have generated two monoclonal antibodies (MAbs 17.9 and A3.2) against Toxoplasma gondii, both of which localize to the dense granules of tachyzoites by immunoelectron microscopy. MAb 17.9 is directed against GRA6, a previously described 32 kDa dense granule protein. MAb A3.2 is directed against a novel 38 kDa dense granule protein, which we refer to as GRA8. GRA8 is released into the parasitophorous vacuole during or shortly after invasion and associates with the periphery of the vacuole. The cDNA sequence encoding GRA8 was determined by screening a T. gondii cDNA expression library with MAb A3.2. The deduced amino acid sequence of GRA8 consists of a polypeptide of 267 amino acids, with no significant homology to any other known protein. The sequence contains an amino terminal signal peptide, three degenerate proline-rich repeats in the central region and a potential transmembrane domain near the carboxy terminus. The most striking feature of GRA8 is its remarkably high proline content (24%).