The results described above indicate that micronemal proteins probably play an important role in Toxoplasma invasion of host cells. Consistent with this idea, we observed previously that Toxoplasma mobilizes MIC2 from its micronemes to its apical surface during attachment to host cells (Carruthers and Sibley, 1997). To investigate the circumstances of MIC2 during penetration, we used double immuno¯uorescence to localize MIC2 in relation to ROP1, a secretory protein that delineates the parasitophorous vacuole. Tightly controlled kinetics allowed us to recreate a time-line representing the progressive release and redistribution of MIC2 during invasion. Consistent with our previous observations (Carruthers and Sibley, 1997), MIC2 was initially secreted at the apical end of parasites that were in intimate contact with the host cell, and this result occurred before secretion of ROP1 into the nascent parasitophorous vacuole (Fig. 5, top row).