Diatoms are unicellular organisms encased by silica-based cell walls that display species-specific structures. Morphogenesis of diatom cell walls is believed to be controlled by a polysaccharide/protein-matrix that remains associated with mature cell walls. Recently, a family of calcium-binding glycoproteins, the frustulins, has been identified as major diatom cell wall component. Here we describe a transformation-based approach to investigate intracellular targeting and function of frustulins. When ε-frustulin from the diatom Navicula pelliculosa is expressed in Cylindrotheca fusiformis, it is correctly targeted into the cell wall. Furthermore, the unique N-terminus of ε-frustulin was properly modified, indicating that C. fusiformis and N. pelliculosa contain homologous frustulin-processing proteases. In a different transformation experiment, a modified version of the Chlorella kessleri hexose/H⁺ symporter bearing a bacterial biotinyl-acceptor domain was expressed in C. fusiformis. The transporter became biotinylated in vivo and was functionally incorporated into the plasma membrane, allowing C. fusiformis to take up ¹⁴C-glucose and ¹⁴C-glucosamine. Stage-specific radioactive labeling with this transformant revealed that secretion of frustulins is strongly enhanced during cell wall development. The data presented in this study demonstrate for the first time functional expression of a membrane protein and correct targeting of a cell wall protein heterologously expressed in a diatom cell.