Diatom cells are encased within a silica-based cell wail (frustule) that serves as armour-like protection for the enclosed protoplast. Maintaining the integrity of the frustule requires a precise coupling between the biogenesis of new frustule components and the cell cycle. Thus far, the molecular mechanisms by which this coupling is achieved are unknown. This study demonstrates that pleuralins (formerly HEPs), a previously characterized family of diatom cell wall proteins, are involved in cell cycle-dependent frustule development. The frustule is made up of two, overlapping half-shells termed the epitheca and hypotheca. Both thecae are morphologically identical, yet immunolocalisation with anti-pleuralin antibodies demonstrates that their protein composition is clearly different. During interphase, pleuralins are associated only with the epitheca, where they are confined to the inner surface of the terminal elements (pleural bands) in the region of overlap with the hypotheca. At cell division, pleuralins also become associated with the newly formed pleural bands of the hypotheca. Remarkably, this process is concomitant with the functional conversion of the parental hypotheca into the epitheca of one of the progeny cells. These results indicate that developmentally controlled association of pleuralins with the frustule is involved in hypotheca-epitheca differentiation, which is a crucial process to ensure proper frustule development.