One of the most efficient anions in enhancing the ability of desferrioxamine (DFO) to remove iron from transferrin in vitro has been shown to be pyrophosphate (PYP). To evaluate the in vivo effect of PYP in hyperferremic mice, the biodistribution of [59Fe]ferric citrate was studied after the i.p. administration of: 1) only saline in the control animals; 2) an aqueous solution of tetrasodium diphosphate (PYP; 40 gm/2 g of b.wt.); 3) desferral (DFO; 12 mg/20 g of b.wt.); and 4) PYP + DFO at the respective dosages shown above. The radioactivity in each organ, blood, urine and feces was measured and referred to as percentage of the injected dose. PYP administered alone acted as a weaker chelator of iron than DFO. The combined administration of DFO and PYP contributed more than DFO or PYP separately, to the increase of urinary excretion of 59Fe and to the significant decrease of the radioiron concentration in liver (.01 less than P less than .05). The above induced changes are not, however, the additive result of the separate effect of DFO and PYP. That observation would suggest that DFO + PYP combined in a unique treatment, interact with iron through a common reaction pathway and that PYP plays in vivo a synergistic role in that interaction. The kind of iron with which DFO + PYP interacts is then suggested to be the transferrin-bound iron located in extracellular spaces of tissues.