The protozoan parasite Toxoplasma gondii is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of T. gondii infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.The apicomplexa are a group of mostly obligate intracellular parasites that are responsible for diseases such as toxoplasmosis, malaria, neosporosis, coccidiosis, and cryptosporidiosis. Host cell invasion is a prerequisite for the establishment and maintenance of infection for these parasites, and although the range of host cell specificity can vary greatly between different apicomplexan species, the machinery they use to invade their host(s) is strikingly conserved. Toxoplasma gondii, the parasite responsible for congenital infections in the developing fetus and for severe neurological complications in immunocompromised individuals, is relatively easy to manipulate genetically, invades virtually all nucleated cells, and, thus, is a good model for studying motility and invasion. Host cell invasion is accompanied by the secretion of parasite proteins from two distinct secretory organelles that are associated with attachment/motility (microneme proteins [MICs]) (23, 26), parasitophorous vacuole biogenesis (rhoptry proteins) (17), and moving junction formation (cooperative role of MICs and rhoptry neck proteins) (2, 27). Exocytosis from dense granules, a third type of specialized secretory granules, occurs during and following the invasion of the host cell and is believed to take part in the maturation of the parasitophorous vacuole, although most of the GRA proteins have not yet been attributed a function (30).The endoplasmic reticulum (ER) is the site of synthesis and the processing of proteins destined for secretion; thus, almost all secretory proteins contain a signal peptide. Most of protein sorting takes place either in the trans-Golgi network (TGN) (or its equivalent) or in immature secretory vesicles beyond the TGN. In T. gondii, soluble foreign reporter proteins, lacking targeting information, are delivered to the parasitophorous vacuole via the dense granules, which are the default route for secreted proteins (22). Similarly to other eukaryotic sorting mechanisms, a system of tyrosine-dependent signals exists in T. gondii that allows for the specific targeting of transmembrane proteins to the rhopt...