Phosphite (Phi, H2PO3-), the reduced form of orthophosphate (Pi, H2PO4-), is broadly used in agriculture as the active compound of fungicides. Nevertheless, under Pi limiting conditions Phi can disturb plant development. The aim of this work was to describe the effects of Phi on the Pi starvation response and the metabolism of plant cells. Morphological and transcriptional studies on Solanum lycopersicum, Nicotiana tabacum and Arabidopsis thaliana confirmed that Phi strongly repressed the Pi starvation response of plant cell cultures and plants. Transport studies on Pi starved tobacco BY-2 cells demonstrated that Phi inhibits the Pi uptake in a competitive manner. In vivo 31P-NMR gave for the first time direct evidence for the intracellular accumulation of Phi in BY-2 plant cells. Furthermore, the in vivo NMR studies allowed following the subcellular distribution of Phi and its effects on plant metabolism. The distribution pattern of Phi depended on the metabolic state of the cells: Pi-deprived cells accumulated Phi predominantly in the cytoplasm, but barely in the vacuolar compartment. In contrast, Pi-preloaded cells accumulated Phi directly into vacuoles. In spite of its distinct cytoplasmic accumulation under Pi deprivation conditions, Phi could not substitute for Pi in plant metabolism. During perfusion with Phi, cell metabolism and energy charge remained unchanged. On the resuply of Pi to Phi perfused cells, Phi was rapidly and selectively exported from the cytoplasm, demonstrating that the BY-2 cells were able to discriminate between Pi and Phi. It appears likely, that the observed exudation of Phi is due to an energy dependent detoxification of Phi.