Hyperthermic spreading depression (HSD) in immature rat hippocampal slices is mediated by Na + /K + -ATPase failure. Here, we test whether depleting intracellular ATP serves as a possible mechanism for HSD genesis. Results indicate that (1) pre-incubation with 3 mM creatine for 3 h failed to prevent hyperthermic spreading depression occurrence; and (2) intracellular ATP concentration doubled during experimental hyperthermia. This study suggests that HSD is not be mediated by depletion of intracellular ATP during hyperthermia.