We examined the Mn(II)-oxidizing ability of the biogenic Mn oxide (BMO) formed in cultures ofa Mn(II)-oxidizing fungus, Acremonium strictum strain KR21-2. The newly formed BMO effectively sequestered dissolved Mn(II) mainly by oxidizing Mn(II) to insoluble Mn under air-equilibrated conditions, and this ability lasted for at least 8 days. Deaerating the BMOs, poisoning them with NaN3, or heating them all readily weakened their Mn(II) oxidation ability, indicating the involvement of enzymatic Mn(II) oxidation. There was no Mn(II)-oxidizing ability observed for mycelia cultivated without Mn(II) or for residual mycelia after the BMO phase was dissolved, suggesting the need for the oxide phase. A sodium dodecyl sulphate-polyacrylamide gel electrophoresis assay demonstrated that the oxide phase embeds the Mn(II) oxidase and thereby maintains the enzymatic activity in BMOs. Freezing at -80 degrees C preserved the Mn(II)-oxidizing ability in BMOs for at least 4 weeks, while lyophilization caused a complete loss of this ability. Based on these results, we propose that fungal Mn oxides supporting Mn(II) oxidase activity are an effective Mn(II)-sequestering material capable of oxidizing Mn(II) continuously from solutions containing no additional nutrients to maintain biological activity.