Using digital imaging microscopy and fluorescent probes, isolated hippocampal CA1 pyramidal neurons of the guinea-pig were used to examine the roles of [Mg2+]o in regulation of [Ca2+]i and [Mg2+]i. Low extracellular Mg ([Mg2+]o) (0.3 mM) significantly increased [Ca2+]i compared to 1.2 and 4.8 mM [Mg2+]o. In contrast, [Mg2+]i levels remained relatively constant, irrespective of alterations of [Mg2+]o. The sustained rise in [Ca2+]i induced by low [Mg2+]o was reduced 70% by 1 microM verapamil and 42% by 1 mM Ni2+, and completely abolished by 5 mM Ni2+. The data suggest that [Mg2+]o regulates [Ca2+]i in hippocampal neurons, probably by modulating Ca2+ entry via voltage-sensitive Ca2+ channels, which may play important roles in epileptogenesis, memory, learning and brain trauma. Furthermore, the results demonstrate that intracellular Mg2+ concentration does not follow passively the concentration of Mg2+ in the extracellular solution.