The cell wall of a diatom is made up of a silica-based scaffold and organic macromolecules. Proteins located in the cell wall are believed to control morphogenesis of the species-specific silica structures of the scaffold. However, data that correlate distinct silica elements and specific proteins within the diatom cell wall have not been reported. Here, the cell wall protein HEP200 (200-kDa HF-extractable protein) from the diatom Cylindrotheca fusiformis is identified and characterized. HEP200 is tightly associated with a substructure of the silica scaffold. It is a member of a new protein family, of which two more members are identified. Each member displays the same bipartite structure. The N-terminal part consists of a variable number of a repeated sequence motif (PSCD domain), whereas the C-terminal part is unique. Immunolocalization experiments revealed the arrangement of different proteins within the cell wall. Frustulins, a previously described group of glycoproteins, constitute the outer coat of the cell wall and exhibit a ubiquitous distribution. In contrast, HEP200 is specifically located at a subset of about six silica strips in intact cell walls, shielded by frustulins. This study therefore identifies a diatom cell wall protein (HEP200) that is associated with a distinct substructure of the silica scaffold.