Three diffusion parameters of nervous tissue, extracellular space (ECS) volume fraction (λ), tortuosity (α) and non-specific uptake (k’) of tetramethylammonium (TMA + ), were studied in the spinal cord of rats during experimental autoimmune encephalomyelitis (EAE). The three parameters were determined in vivo from concentration-time profiles of TMA+ using ion-selective microelectrodes. EAE was induced by injection of guinea-pig myelin basic protein (MBP), which resulted in typical morphological changes in the CNS tissue, namely inflammatory reaction, astrogliosis, blood-brain barrier (BBB) damage and paralysis. EAE was accompanied by a statistically significant increase of a (mean±S.E.M.) in the dorsal horn from 0.21±0.01 to 0.28±0.02, in the intermediate region from 0.22±0.01 to 0.33±0.02, in the ventral horn from 0.23±0.01 to 0.47±0.02 and in white matter from 0.18±0.03 to 0.30±0.03. There were significant decreases in tortuosity in the dorsal horn and in the intermediate region and decreases in non-specific uptake in the intermediate region and in the ventral horn. Although the inflammatory reaction and the astrogliosis preceded and greatly outlasted the neurological symptoms, the BBB damage had a similar time course. Moreover, there was a close correlation between the changes in extracellular space diffusion parameters and the manifestation of neurological signs. We suggest that the expansion of the extracellular space alters the diffusion properties in the spinal cord. This may affect synaptic as well as non-synaptic transmission, intercellular communication and recovery from acute EAE, and may contribute to the manifestation of neurological signs in EAE rats.