Tenascin-R (TN-R), a large extracellular glycoprotein, is an important component of the adult brain's extracellular matrix (ECM); tenascin-C (TN-C) is expressed mainly during early development, while human natural killer 1 (HNK-1) is a sulphated carbohydrate epitope that attaches to these molecules, modifying their adhesive properties. To assess their influence on extracellular space (ECS) volume and geometry, we used the real-time iontophoretic method to measure ECS volume fraction α and tortuosity λ, and diffusion-weighted magnetic resonance imaging (MRI) to measure the apparent diffusion coefficient of water (ADCW). Measurements were performed in vivo in the cortex and CA1 hippocampal region of TN-R-, TN-C- and HNK-1 sulphotransferase (ST)-deficient adult mice and their wild-type littermate controls. In both cortex and hippocampus, the lack of TN-R or HNK-1 sulphotransferase resulted in a significant decrease in α and λ. Compared with controls, α in TN-R–/– and ST–/– mice decreased by 22–26% and 9–15%, respectively. MRI measurements revealed a decreased ADCW in the cortex, hippocampus and thalamus. ADCW reflected the changes in α; the decrease in λ indicated fewer diffusion obstacles in the ECS, presumably due to a decreased macromolecular content. No significant changes were found in TN-C–/– animals. We conclude that in TN-R–/– and ST–/– mice, which show morphological, electrophysiological and behavioural abnormalities, the ECS is reduced and its geometry altered. TN-R, as an important component of the ECM, appears to maintain an optimal distance between cells. The altered diffusion of neuroactive substances in the brain will inevitably affect extrasynaptic transmission, neuron–glia interactions and synaptic efficacy.