The reaction mechanism of p-nitrophenylphosphatase (PNNPase) of SR was elucidated by analyzing its partial reaction using radioactive PNPP as substrate. The following results were obtained: 1. Phosphoprotein was produced by the reaction of SR with PNPP at an alkaline pH and in the presence of a high concentration of Ca2+ ions, where it decomposed very slowly. the amount of phosphoprotein reached 0.2 mol/105g in the presence of 10 mM CaCl2 and 1 mM MgCl2 at pH 8.75 and 15°C. The rate of phosphoprotein formation of PNPPase was much lower than that of ATPase. The amount of phosphoprotein decreased, while the rate of PNP liberation increased, immediately after the pH and/of free Ca2+ concentration was reduced. 2. When ADP reacted with 32P-labelled phosphoprotein produced from (32P)PNPP, the amount of 32P-labelled phosphoprotein decreased rapidly and (γ-32P)ATP was formed almost stoichiometrically. The pH stability and hydroxylamine sensitivity of phosphoprotein of PNPPase denatured with TCA were the same as those of phosphoprotein of ATPase. 3. The phosphorylation reaction was started by adding 32P)PNPP and stopped by dilution with a 40-fold volume of the reaction mixture containing unlabelled PNPP, then the time course of the subsequent decrease in the amount of phosphoprotein was measured. The time course was biphasic, indicating the existence of two kinds of phosphoprotein. In the presence of 5 mM CaCl2 and 5 mM MgCl2 at pH 8.0, the decay constants were about 2 min−1 and 0.1 min−1, respectively. The ratio of the amounts of fast and slowly decomposing components of the phosphoprotein was about 1 : 4. The decay rates and the ratio of the two components were unaffected by preincubation of SR with PNP. The decay constant of the slow phase increased when the Mg2+ concentration increased or the Ca2+ concentration decreased. However, the decay constant of the rapid phase was virtually unaffected by changing concentrations of Mg2+ and Ca2+ ions. 4. The time course of PNP liberation was resolved into two phases consisting of a fast initial phase (burst) and a slow steady phase. The burst size of PNP liberation was about 0.5 mol/105, which was larger than the amount of phosphoprotein under the same conditions, i.e., 0.25 mol/105 g.