Diffusion-weighted imaging (DWI) is frequently used to diagnose stroke. However, the origin of the observed reduction in the apparent diffusion coefficient (ADC) in the acute phase following ischemia is not well understood. Although cell swelling is considered to play an important role, it is unclear whether this can completely explain the large ADC decrease. We developed a method to induce in neonatal rat hippocampal slices both osmotic perturbations, which lead to cell swelling, and oxygen/glucose deprivation (OGD), which simulates ischemia. A perfusion system was used to provide the hippocampal slices with nutrients and oxygen to maintain slice viability, which was verified with the use of fluorescent dyes (live/dead staining). Upon induction of OGD, the ADC decreased to 57% of the initial value within 2 hr. The ADC reduction cannot fully be explained by changes due to cell swelling, since these led only to a maximum decrease of 83%. Therefore, in addition to cell swelling, other changes must contribute significantly to the ADC reduction.