AbstractThe effect of lipids on protoheme ferrolyase, which combines ferrous iron with protoporphyrin, was investigated. The enzyme extracted with 1% Na cholate from acetone‐dried powder of chicken erythrocyte stroma showed high enzymic activity, while that extracted with 0.4 M KCl showed little activity. The former contained lipids, the main components of which were lecithin and phosphatidylethanolamine, whereas the latter contained little lipid. Crude lipids of several sources restored the enzyme activity of 0.4 M KCl extracts. The activating effects of purified lipids, especially phospholipids which showed the strongest activation, were further examined. Phospholipids were divided into three groups: the choline‐containing group, lecithin and sphingomyelin, was inhibitory or slightly accelerative on heme synthesis; the acidic phospholipids, namely phosphatidylethanolamine, cardiolipin, phosphatidic acid and phosphatidylinositol, were strong activators and the intensity of activation was in the order of the acidity of the phospholipids; and the lysophospholipids, namely lysolecithin, lysophosphatidylethanolamine and sphingosylphosphorylcholine, activated the heme synthesis most effectively. In the presence of cholate, choline‐containing lipids were highly effective, while acidic phospholipids were inhibitory and lysophospholipids were neutral. Palmitic acid showed slight stimulative effect. Tripalmitin was neutral or inhibitory. Anionic, cationic and neutral synthetic detergents were slightly stimulative in low concentration and inhibitory in high concentration. An activation mechanism of phospholipids was proposed in which the hydrophilic anionic part of lipid in the lipoprotein enzyme molecule attracts ferrous iron. After being stripped of solvation water, the ferrous iron is transferred to the hydrophobic part of the enzyme molecule to be inserted into porphyrin.