Mg2+ plays an important role in biological functions, similar to that of Ca2+. In terms of neural activity, it is well known that Mg2+ blocks the NMDA receptor. However, the relationship between Mg2+ and neural function has not been well understood. We have investigated the effect of low extracellular Mg2+ concentration ([Mg2+]o) on neural activity in rat cortical and hippocampal neurons by using microelectrode array (MEA) measurements and glutamate measurements, with an enzyme modified MEA-based multi-array sensor. In this study, we investigated the effects of low [Mg2+]o on intracellular Ca2+ concentration ([Ca2+]i) using a confocal laser microscope and a flow cytometer with a fluorescence probe. The results indicate that low [Mg2+]o has an effect on neural activity. The responses of cortical and hippocampal neurons to low [Mg2+]o differed in the developmental period. The results suggest that hippocampal neurons are more sensitive to [Mg2+ than cortical neurons. The glutamate receptor distributions in the cortex and hippocampus may be different. Further investigation is required to understand the mechanisms of the Mg2+ effect on neural activity.