During ischemia, the transport of molecules in the extracellular space (ECS) is obstructed in comparison with healthy brain tissue, but the cause is unknown. Extracellular tortuosity (lambda), normally 1.6, increases to 1.9 in ischemic thick brain slices (1000 microm), but drops to 1.5 when 70,000 Mr dextran (dex70) is added to the tissue as a background macromolecule. We hypothesized that the ischemic increase in lambda arises from diffusion delays in newly formed dead-space microdomains of the ECS. Accordingly, lambda decreases when dead-space diffusion is eliminated by trapping dex70 in these microdomains. We tested our hypothesis by analyzing the diffusion of several molecules in neocortical slices. First we showed that diffusion of fluorescent dex70 in thick slices declined over time, indicating the entrapment of background macromolecules. Next, we measured diffusion of tetramethylammonium (TMA+) (74 Mr) to show that the reduction of lambda depended on the size of the background macromolecule. The synthetic polymer, 40,000 Mr polyvinylpyrrolidone, reduced lambda in thick slices, whereas 10,000 Mr dextran did not. The dex70 was also effective in normoxic slices (400 microm) after hypoosmotic stress altered the ECS to mimic ischemia. Finally, the dex70 effect was confirmed independently of TMA+ using fluorescent 3000 Mr dextran as a diffusion marker in thick slices: lambda decreased from 3.29 to 2.44. Taken together, these data support our hypothesis and offer a novel explanation for the origin of the large lambda observed in ischemic brain. A semiquantitative model of dead-space diffusion corroborates this new interpretation of lambda.