Using ATP directly synthetical method with ADP and radiophosphate, the effects of azide and inorganic phosphate on the ATP synthesis of F₁F_O-ATPase from pig heart mitochondria are investigated. It is found that P_i not only takes an active part in ATP synthesis as a substrate, but also has an inhibition effect on ATP synthesis of F₁F_O-ATPase. In the presence of 1 mmol/L ADP the inhibition increases with the increase of P_i concentration from 0.01 mmol/L to 10 mmol/L. The viewpoint is different from that azide only inhibites ATP hydrolysis but not affects the ATP synthesis at low concentration (＜1 mmol/L). The results indicate that 0.1 mmol/L azide apparently activates ATP synthesis of F₁F_O-ATPase. The activation is negatively related to the concentration of P_i in the reaction system. When the concentration of P_i is kept at 0.1 mmol/L, with the increase of azide concentration, the apparent activation degree of ATP synthesis by azide is changed. When the concentrations of azide and P_i are equal, the apparent activation by azide is the biggest. The apparent inhibition of ATP synthesis by azide would not begin until the concentration of azide reaches near 0.5 mmol/L. After the concentration of azide is higher than 1 mmol/L, the uncoupling of F₁F_O-ATPase appears. The mechanism of the roles of azide and P_i is discussed.