Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and tortuosity, leading to a modified diffusion equation that represents the transport behavior of many molecules. Early studies used radiolabeled tracers, while current methods include real-time iontophoresis (RTI) for small ions and integrative optical imaging (IOI) for fluorescent macromolecules. Theoretical models and simulations explore the influence of ECS geometry, dead-space microdomains, extracellular matrix, and macromolecule interactions. Experimental studies with RTI show that the ECS volume fraction is typically about 20%, and the tortuosity is about 1.6, with regional variations. These properties change during development and aging. Diffusion properties have been characterized in various interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. These studies enhance our understanding of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment, which is crucial for understanding extrasynaptic volume transmission and drug delivery to the brain.Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and tortuosity, leading to a modified diffusion equation that represents the transport behavior of many molecules. Early studies used radiolabeled tracers, while current methods include real-time iontophoresis (RTI) for small ions and integrative optical imaging (IOI) for fluorescent macromolecules. Theoretical models and simulations explore the influence of ECS geometry, dead-space microdomains, extracellular matrix, and macromolecule interactions. Experimental studies with RTI show that the ECS volume fraction is typically about 20%, and the tortuosity is about 1.6, with regional variations. These properties change during development and aging. Diffusion properties have been characterized in various interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. These studies enhance our understanding of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment, which is crucial for understanding extrasynaptic volume transmission and drug delivery to the brain.