A second heavier organic phase of tetravalent plutonium nitrate (plutonium third phase) was investigated on the formation requirement and the accumulation process in mixer-settlers, with 30% tributylphosphate (TBP) in n-dodecane. The amount of TBP of the third phase, which split from the organic phase, was twice as great as that of the source organic phase and was saturated with plutonium and nitric acid. The plutonium and nitric acid in the third phase were regulated in concentration by a relation of the solubility product, which gives the minimum of plutonium and nitric acid necessary to form the third phase. From the minimum of nitric acid, 185 g/ℓ of plutonium was estimated as the maximum concentration in the third phase, which agreed with the experimental data in another report. The accumulation process of the third phase in mixer-settlers was simulated with the use of a distribution relation of plutonium between the third phase and ∼30% TBP organic phase. The simulated results agreed well with the actual results of the plutonium concentration, the volume, and the distribution profile in stages, on the assumption that 5% of the plutonium third phase formed in the organic phase splits to the plutonium third phase and stays at the aqueous/organic interface of mixer-settlers.