The aim of this study was to examine the effect of the archetypal pro-inflammatory cytokine, interleukin-1beta (IL-1β), on high-energy phosphate levels within an ex vivo rat organotypic hippocampal-slice culture (OHSC) preparation using phosphorus ( 31P) magnetic resonance spectroscopy (MRS). Intrastriatal microinjection of IL-1β induces a chronic reduction in the apparent diffusion coefficient (ADC) of tissue water, which may be indicative of metabolic failure as established by in vivo models of acute cerebral ischaemia. The OHSC preparation enables examination of the effects of IL-1β on brain parenchyma per se, independent of the potentially confounding effects encountered in vivo such as perfusion changes, blood-brain barrier (BBB) breakdown and leukocyte recruitment. 31P MRS is a technique that can detect multiple high-energy phosphate metabolites within a sample non-invasively. Here, for the first time, we characterise the energy metabolism of OHSCs using 31P MRS and demonstrate that IL-1β does not compromise high-energy phosphate metabolism. Thus, the chronic reduction in ADC observed in vivo is unlikely to be a consequence of metabolic failure.