Serotonin (5-hydroxytryptamine) is an important neurotransmitter in organisms, regulating for example mood, thermoregulation, also functioning of muscles and heart. Serotonin_1A receptors (5-HT_1A receptor) belongs to the superfamily of seven-transmembrane G-protein coupled receptors (GPCR) and is widely distibuted in brain, the highest densities are found in hippocampus, septum, amygdala and cortical limbic areas. 5-HT_1A receptors couple to G_i/o-proteins, resulting in decreased levels of intracellular cyclic adenosine monophosphate. Signalling through 5-HT_1A receptor has been linked to mood, learning, memory, eating and sexual behavior. It has been generally accepted that Mg²⁺-ions participate in signal transduction via GPCR-s, where Mg²⁺-ions are required to achieve high affinity agonist binding and regulate functions of G proteins. Other ions of this group (Mn²⁺, Al³⁺, Zn²⁺ etc) have been considered to act on different biochemical pathways and on toxic doses changing the pattern of reaction via alterating, inhibiting or activating different biochemical reactions. The modulatory effect of Mn²⁺ has been found also in the case of 5-HT_1A receptors. Herewith we have found that effects of Mn²⁺ and Mg²⁺-on regulation of agonist binding to 5-HT_1A receptors can be very similar, but also different, depending on the region of rat brain studied. These ions did not alter binding properties of 5-HT_1A antagonist [³H]WAY-100635, but Mn²⁺-ions enhanced the ability of serotonergic agonists to compete with [³H]WAY-100635 in hippocampal membranes compared to Mg²⁺-ions. Mn²⁺-ions were more effective in enhancing of the binding of 5-HT_1A agonist [³H]8-OH-DPAT, but also in changing the abilities of nucleotides to turn the receptors into low-affinity agonist binding state. The biggest differences was found in hippocampal membranes, whereas effects in cortical membranes and in membranes prepared of Sf9-cells (with expressed 5-HT_1A receptor and Gi-protein) were considerably smaller. No ion-specific effect was seen in Sf9 membranes expressing 5-HT_1A receptor alone or 5-HT_1A receptor with Gs-proteins. These results indicate that signal transduction via 5-HT_1A receptor in different parts of brain is mediated by different regulation systems, which also vary by their sensitivity towards Mn²⁺– and Mg²⁺-ions.