The role of calcium ions in the phospholipid hydrolysis by phospholipase D was studied. It was shown that the enzyme does not split egg lecithine in the absence of Ca2+. In the presence of Ca2+ the reaction occurs via different routes, depending on the type of initiation of the reaction. The optimal concentrations of Ca2+ necessary for activation of phospholipase D are different in the systems activated by various treatments (organic solvents, detergents and solid adsorbents). Optimal concentrations of Ca2+ for the hydrolysis and methanolysis catalyzed by phospholipase D are also different. It was found that the need for Ca2+ and their optimal concentrations are determined by the state of phospholipids at the substrate phase. The data suggest that the enzymatic hydrolysis may occur in the absence of Ca2+. Thus, Ca2+-induced activation is merely an alternative pathway of catalytically active conformation of lypolytic enzymes.