A new class of inositol phosphates containing energy-rich pyrophosphoryl residues has been characterized. D/L-1-Diphosphoinositol pentakisphosphate(s) and D/L-bis-(1,4)-diphosphoinositol tetrakisphosphate(s) are present as soluble ionic species in the cytosol of amoebae (Dictyostelium discoideum) at concentrations in the range of 0.05-0.25 mM. These compounds are rapidly metabolized in intact cells and can be synthesized in cell lysates from myo-inositol hexakisphosphate in the presence of ATP. Their phosphomonoester groups have predicted C-O-P bond energies of between 3.3 and 4 kcal mol-1; however, the bond energies of the P-O-P links in their diphosphate moieties are 6.6 kcal mol-1 and hence similar to the equivalent bonds in ADP, indicating a potential role for these compounds as phosphate donors in phosphotransferase reactions. Compounds with similar chromatographic properties are found in a variety of mammalian cell types.