To further examine the potential clinical usefulness of the hexadentate phenolic aminocarboxylate iron chelator N, N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) for the chronic treatment of transfusional iron overload, we performed a subchronic toxicity study of the HBED monosodium salt in rodents and have evaluated the iron excretion in primates induced by HBED. The HBED-induced iron excretion was determined for the monohydrochloride dihydrate that was first dissolved in a 0.1-mmol/L sodium phosphate buffer at pH 7.6 and administered to the primates either orally (PO) at a dose of 324 micromol/kg (149.3 mg/kg, n = 5), subcutaneously (sc) at a dose of 81 micromol/kg (37.3 mg/kg, n = 5), sc at 324 micromol/kg (n = 5), and sc at 162 micromol/kg (74.7 mg/kg) for 2 consecutive days for a total dose of 324 micromol/kg (n = 3). In addition, the monosodium salt of HBED in saline was administered to the monkeys sc at a single dose of 150 micromol/kg (64.9 mg/kg, n = 5) or at a dose of 75 micromol/kg every other day for three doses, for a total dose of 225 micromol/kg (n = 4). For comparative purposes, we have also administered deferoxamine (DFO) PO and sc in aqueous solution at a dose of 300 micromol/kg (200 mg/kg). In the iron-loaded Cebus apella monkey, whereas the PO administration of DFO or HBED even at a dose of 300 to 324 micromol/kg was ineffective, the sc injection of HBED in buffer or its monosodium salt, 75 to 324 micromol/kg, produced a net iron excretion that was nearly three times that observed after similar doses of sc DFO. In patients with transfusional iron overload, sc injections of HBED may provide a much needed alternative to the use of prolonged parenteral infusions of DFO. Note: After the publication of our previous paper (Blood, 91:1446, 1998) and the completion of the studies described here, it was discovered that the HBED obtained from Strem Chemical Co (Newburyport, MA) that was labeled and sold as a dihydrochloride dihydrate was in fact the monohydrochloride dihydrate. Therefore, the actual administered doses were 81, 162, or 324 micromol/kg; not 75, 150, or 300 micromol/kg as was previously reported. The new data have been recalculated accordingly, and the data from our earlier study, corrected where applicable, are shown in parentheses.