Two intracellular pools of soluble polyphosphate were identified by in vivo 31P NMR spectroscopy in the cyanobacterium Synechocystis sp. strain PCC 6308. Polyphosphate was present in the cells after growth in sulfur-limited media containing excess phosphate. The presence of polyphosphate was confirmed by transmission electron microscopy and chemical analysis. 31P NMR spectroscopy of whole cells treated with EDTA revealed two pools of mobile polyphosphate. A downfield shift and narrowing of part of the broad polyphosphate resonance was observed after EDTA treatment, suggesting that EDTA binds metal ions normally associated with some of the polyphosphate. Phosphate, but not polyphosphate, leaked out of the cells after this treatment. Addition of magnesium ions caused the downfield shift in the polyphosphate resonance to move back toward its original value. These data show that only part of the cation-complexed polyphosphate is accessible to the added EDTA and suggest that there are two internal fractions of NMR-visible polyphosphate in the cells, only one of which loses its associated cations to EDTA. Spheroplast formation showed that polyphosphate was not present in the periplasm of the cells.