In order to investigate the cellular system of the freshwater sponge, Ephydatia fluviatilis, we isolated a molecular marker for the most prominent cell type, the choanocyte. After feeding sponge with fluorescent beads, fluorescent-labeled choanocytes were collected by fluorescence activated cell sorting (FACS). By protein profiling choanocyte and archeocyte (stem cell)-rich fractions, proteins characteristic of choanocyte were identified. The partial amino-acid sequence of one of the proteins characteristic of choanocyte matches the deduced amino-acid sequence of sponge expression tag (EST) clones and mouse annexin VII. These EST clones overlap and encode a protein, designated Ef annexin, which includes four annexin domains. Whole mount in situ hybridization shows Ef annexin expression in chamber-forming choanocytes in 7-day-old sponge, leading us to conclude that Ef annexin can be used as a choanocyte marker. In the early development stage, Ef annexin expression can be detected in both large single cells, characteristic of archeocytes, and cells forming 2-, 4- and multiple-cell clusters. These results indicate that Ef annexin is initially expressed in the choanocyte-committed archeocyte which then undergoes several mitotic cell divisions to form a choanocyte chamber. This suggests that the single choanocyte chamber essentially originates from a single archeocyte.