Algae with secondary plastids such as diatoms maintain two different eukaryotic cytoplasms. One of them, the so-called periplastidal compartment (PPC), is the naturally minimized cytoplasm of a eukaryotic endosymbiont. In order to investigate the protein composition of the PPC of diatoms, we applied knowledge of the targeting signals of PPC-directed proteins in searches of the genome data for proteins acting in the PPC and proved their in vivo localization via expressing green fluorescent protein (GFP) fusions. Our investigation increased the knowledge of the protein content of the PPC approximately 3-fold and thereby indicated that this narrow compartment was functionally reduced to some important cellular functions with nearly no housekeeping biochemical pathways.