Using digital imaging microscopy and fluorescent probes, isolated hippocampal CAI 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 μM verapamil and 42%v 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.