To investigate the influence of osmolarity on epileptiform activity in immature hippocampus, extracellular field potentials were recorded in the CA3 region of hippocampal slices of immature (postnatal day 4–7) Wistar rats. The diffusion parameters of the extracellular space were determined by the real-time tetramethylammonium (TMA) iontophoretic method using ion-selective microelectrodes in immature hippocampal slices and showed a lack of diffusion anisotropy, a tortuosity of about 1.39 and a volume fraction a of 0.41 ± 0.01 (n=10 slices). A reduction in osmolarity by 100 mOsm induced a decrease in a to 0.17 ± 0.02 (n=4 slices) and increased the frequency of epileptiform activity elicited in 10–50 mM 4-AP containing low-Mg ²⁺ solution by 39 ± 9.2 % (n=16 slices). Hypoosmolar conditions also enhanced the incidence rate of epileptiform activity observed in low-Mg²⁺ solution from 0.13 to 0.47 (n=13 and 19 slices, respectively). Hyperosmolar solutions were without effect on epileptiform activity. In summary, these results demonstrate that hypoosmolar conditions increased excitability and seizure susceptibility in immature hippocampal slices, suggesting an involvement of the larger a in suppression of seizures. Supported by DFG grants Ki 835/2-1 to WK, Lu 375/4 to HJL and grants AVOZ 50390512, 1M0021620803 to ES