Freshly isolated protein phosphatase 2A (PP2A) was highly active as to the dephosphorylation of protein substrates, but lost most of its spontaneous activity on prolonged storage, and was converted to a latent form requiring Mn²⁺ or Co²⁺ ions for activity. In this report, we show that the latent form of PP2A can be activated by the Fe²⁺/ascorbate system. Activation of the phosphatase required both Fe²⁺ ions and ascorbate, and the level of activation was dependent on the concentrations of both Fe²⁺ ions and ascorbate. Both the holoenzyme and catalytic subunit of phosphatase 2A could be activated by the Fe²⁺/ ascorbate system, indicating that direct modulation of the catalytic subunit of the phosphatase by the ^Fe2+/ascorbate system may cause this activation. Several common divalent metal ions, including Ca²⁺, Mg²⁺, Cu²⁺, Zn²⁺, and Ni²⁺ ions, cannot cooperate with ascorbate to activate the phosphatase. Dithiothreitol, a SH-containing reducing agent, could replace ascorbate in the Fe²⁺/ascorbate system to activate the phosphatase, whereas H₂O₂, a strong oxidizer, significantly diminished the phosphatase activation by the Fe²⁺/ascorbate system. The results indicate that iron ions stabilized in the +2 state by reducing agents can activate the phosphatase. Overall, the present study provides initial biochemical evidence suggesting that Fe²⁺ could be a biologically important metal ion cofactor responsible for PP2A activation.