The PII protein is an evolutionary highly conserved regulatory protein from bacteria to higher plants. In bacteria it modulates the activity of several enzymes, transporters and regulatory factors by interacting with them and thereby regulating important metabolic hubs like carbon/nitrogen homeostasis. More than two decades ago the PII protein was characterized for the first time in plants, but its physiological role is still not sufficiently resolved. To gain more insights into the function of this protein, we investigated the interaction behaviour of AtPII with candidate proteins by BiFC and FRET/FLIM in planta and with GFP/RFP traps in vitro. In the course of these studies we found that AtPII interacts in chloroplasts with itself as well as with known interactors like NAGK in dot-like aggregates, which we named PII foci. In these novel protein aggregates AtPII interacts also with yet unknown partners, which are known to be involved in plastidic protein degradation. Further studies revealed that the C-terminal part of AtPII is crucial for the formation of PII foci. Altogether, the presented results indicate a novel mode of interaction for PII proteins with other proteins in plants, which may be a new starting point for the elucidation of physiological functions of PII proteins in plants.