Biological membranes show uneven distribution of different types of lipids in the inner and outer layers, resulting in transverse asymmetric membranes. The treatment of the erythrocyte membrane with the enzyme phospholipase confirmed the asymmetric nature of the lipid bilayer. The enzyme hydrolyzes lipids into fatty acids and hydrophilic groups. The phospholipase acts only on the outer layer of the membrane, while the inner layer remains intact. The phospholipase treatment resulted in 80% degradation of total membrane phosphatidylcholine (PC), while only 20% of phosphatidylserine (PS) and phosphatidylethanolamine (PE). The experimental result suggested that in erythrocyte membranes, the majority of PC is present in the outer layer, whereas the PS and PE are prominently located in the inner layer.

The distribution of lipids in each layer of the bilayer happens according to their functional needs as the inner and the outer layer of a lipid bilayer face different environments. For example, negatively charged PS is necessary for the functioning of intracellular enzymes such as protein kinase C. Phosphatidylinositol (PI) is also present in the inner layer; when phosphorylated, it binds and localizes various cytosolic proteins involved in cell signaling.

Some lipids like sphingolipids and cholesterol are known to form microdomains in the outer layer which looks like small islands in the membrane sea called the lipid rafts. These microdomains are often associated with the glycoinositolphospholipids or GPI-coupled proteins, generating lateral membrane asymmetry. Apart from the lipids, proteins, and carbohydrates also contribute to lipid asymmetry. The inner and outer layer is associated with different proteins, whereas carbohydrates, in the form of glycoproteins and glycolipids, are always associated with the outer layer, further adding to the membrane asymmetry.