Microsporidia are obligate intracellular pathogens which enter host cells by the discharge of a hollow tube through which the sporoplasma is extruded into the host cell. Since this invasion mechanism is very different from common entry strategies, the formation of the parasitophorous vacuole (PV) in Encephalitozoon species is likely to be distinct from known principles. We investigated the origin of the nascent Encephalitozoon cuniculi PV membrane with the aid of fluorescent lipid probes. When Bodipy 500/510-C 12 -HPC-labeled spores were used for infection, the emerging PV membrane was unlabeled, suggesting that sporoplasma-derived lipids do not significantly contribute to the formation of the PV membrane. In contrast, when raft and nonraft microdomains of the host cell plasma membrane were selectively labeled with DiIC 16 and Speedy DiO, both tracers were detectable in the nascent PV membrane shortly after infection, indicating that the bulk lipids of the PV membrane are host cell derived. Time-lapse fluorescence microscopy revealed that the formation of the PV membrane is a fast event (<1.3 s), which occurred simultaneously with the extrusion of the sporoplasma. The portion of the discharged tube which is in contact with the host cell was found to be coated with labeled host cell lipids, which might be an indication for a plasma membrane invagination at the contact site. To investigate the presence of pores in the E. cuniculi PV membrane, we microinjected fluorescent dyes of different sizes into infected host cells. A 0.5-kDa dextran as well as 0.8-to 1.1-kDa peptides could rapidly enter the PV, while a 10-kDa dextran was stably excluded from the PV lumen, indicating that the PV membrane possesses pores with an exclusion size of <10 kDa, which should allow metabolite exchange.Microsporidia are obligate intracellular microorganisms which are early branched fungi as revealed by comparative genome analysis (11,12,14). They were recognized in the past years as emerging pathogens, particularly for immunocompromised patients. Three out of the four most common human microsporidian pathogens belong to the genus Encephalitozoon, namely, E. cuniculi, E. hellem, and E. intestinalis. While most microsporidia replicate inside the host cell cytosol, the members of this genus reside inside a membrane-bound vacuolar compartment termed the parasitophorous vacuole (PV). E. cuniculi has become a model organism for this genus, since it can be easily cultured in a variety of host cells and its genome is completely sequenced. The E. cuniculi genome is, at 2.9 Mb, the smallest eukaryotic genome sequenced so far and displays a high degree of compactness and an extreme loss of biosynthetic pathways (13, 15).The intracellular life cycle of E. cuniculi includes the replication of meronts and the subsequent differentiation into sporonts and spores, which are released from the dying host cell (5). The infectious stage is the spore, which contains a unique invasion apparatus to infect host cells. Inside the spore, a long, coiled hollow tub...