Apo horse spleen ferritin (apo HoSF) was reconstituted to various core sizes (100-3500 Fe3+/HoSF) by depositing Fe(OH)3 within the hollow HoSF interior by air oxidation of Fe2+, Fe2+ and phosphate (P(i)) were then added anaerobically at a 1:4 ratio, and both Fe2+ and P(i) were incorporated into the HoSF cores. The resulting P(i) layer consisted of Fe2+ and P(i) at about a 1:3 ratio which is strongly attached to the reconstituted ferritin mineral core surface and is stable even after air oxidation of the bound Fe2+. The total amount of Fe2+ and P(i) bound to the iron core surface increases as the core volume increases up to a maximum near 2500 iron atoms, above which the size of the P(i) layer decreases with increasing core size. Mossbauer spectroscopic measurements of the P(i)- reconstituted HoSF cores using 57Fe2+ show that 57Fe3+ is the major species present under anaerobic conditions. This result suggests that the incoming 57Fe2+ undergoes an internal redox reaction to form 57Fe3+ during the formation of the (P)i layer. Addition of bipyridine removes the 57Fe3+ bound in the P(i) layer as [57Fe(bipy)3]2+, showing that the bound 57Fe2+ has not undergone irreversible oxidation. This result is related to previous studies showing that 57Fe2+ bound to native core is reversibly oxidized under anaerobic conditions in native holo bacterial and HoSF ferritins. Attempts to bury the P(i) layer of native or reconstituted HoSF by adding 1000 additional iron atoms were not successful, suggesting that after its formation, the P(i) layer 'floats' on the developing iron mineral core.