The chapter discusses the molecular basis of bacterial outer membrane permeability, focusing on the structure and function of the outer membrane in Gram-negative bacteria. The outer membrane, which is a trilaminar structure located outside the peptidoglycan layer, plays a crucial role in protecting the cell from host defense factors and antibiotics. It also contributes to the hydrophilicity of the bacterial surface, aiding in evading phagocytosis and complement resistance. The outer membrane is composed of phospholipids, lipopolysaccharides (LPS), and proteins. LPS, a characteristic component, has a complex structure with six or seven fatty acid chains and a hydrophobic membrane-anchoring region. The proteins in the outer membrane include porins, which allow the passage of small hydrophilic molecules, and specific transport proteins like LamB for maltose and Ttx for nucleosides. Porins, the main channel proteins, are rich in β-sheet structure and form ordered hexagonal arrays. They have a strong affinity for LPS and can be voltage-controlled. The permeability of porin channels is influenced by solute size, charge, and hydrophobicity. For example, negatively charged solutes diffuse more slowly through the channels compared to uncharged ones, and hydrophobic solutes are generally excluded. The chapter also discusses the methods used to study outer membrane permeability, including liposome swelling assays and intact cell studies. These methods help in understanding the molecular aspects of permeability and the interplay between the outer and inner membranes in determining transport processes.The chapter discusses the molecular basis of bacterial outer membrane permeability, focusing on the structure and function of the outer membrane in Gram-negative bacteria. The outer membrane, which is a trilaminar structure located outside the peptidoglycan layer, plays a crucial role in protecting the cell from host defense factors and antibiotics. It also contributes to the hydrophilicity of the bacterial surface, aiding in evading phagocytosis and complement resistance. The outer membrane is composed of phospholipids, lipopolysaccharides (LPS), and proteins. LPS, a characteristic component, has a complex structure with six or seven fatty acid chains and a hydrophobic membrane-anchoring region. The proteins in the outer membrane include porins, which allow the passage of small hydrophilic molecules, and specific transport proteins like LamB for maltose and Ttx for nucleosides. Porins, the main channel proteins, are rich in β-sheet structure and form ordered hexagonal arrays. They have a strong affinity for LPS and can be voltage-controlled. The permeability of porin channels is influenced by solute size, charge, and hydrophobicity. For example, negatively charged solutes diffuse more slowly through the channels compared to uncharged ones, and hydrophobic solutes are generally excluded. The chapter also discusses the methods used to study outer membrane permeability, including liposome swelling assays and intact cell studies. These methods help in understanding the molecular aspects of permeability and the interplay between the outer and inner membranes in determining transport processes.