Mitochondria purified from Lentinus edodes were dialyzed against 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM ethylenediaminetetraacetic acid (EDTA) or 10 mM 1,10-phenanthroline (o-Phe) for 48 hs in order to remove metal ions from the mitochondria. Removal of non-heme iron ions from mitochondria by dialysis against 10 mM EDTA led to a 52% inactivation of the native enzyme. Enzyme activity was 93% reactivated by addition of 0.5 mM $Fe^{2+}$ and, to a lesser extent, by addition of 1.0 mM $Mg^{2+}$. $Fe^{3+}$ ion had no significant influence. The effect of 0.5 mM $Fe^{2+}$ in the presence of 1.0 mM $Mg^{2+}$ was similar to that of 0.5 mM $Fe^{2+}$ in the absence of $Mg^{2+}$. $Fe^{2+}$ contributes to enzyme activity independently of $Mg^{2+}$. The enzyme dialyzed against 10 mM o-Phe showed the similar results. $Fe^{2+}$ is required for the activity of mitochondrial ATP synthase in L. edodes. The $K_m$ value of the enzyme was 1.43 mM for ADP as a substrate. and was 0.48 mM in the presence of 0.5 mM $Fe^{2+}$.