We investigated species difference in binding of major neurotransmitters and intracellular second messengers in the gerbil brain and the rat brain using receptor autoradiography. [3H]Phorbol 12,13-dibutyrate (PDBu), [3H]inositol 1,4,5-trisphosphate (IP3), [3H]PN200-110, [3H]muscimol, [3H]MK-801, [3H]cyclohexyladenosine (CHA),and [3H]quinuclidinyl benzilate (QNB) were used to label protein kinase C, IP3 receptor, L-type calcium channel, gamma-aminobutyric acidA (GABAA) receptor, N-methyl-D-aspartate (NMDA) receptor, adenosine A1 receptor, and muscarinic cholinergic receptor, respectively. Autoradiographic distributions of the bindings of most neurotransmitters and second messengers were particularly found in the limbic system and basal ganglia in both gerbil and rat brains. However, marked differences in these bindings between the gerbil brain and the rat brain were also recognized in the above regions. In particular, among 7 ligands used, the gerbil had high [3H]PDBu and [3H]CHA binding sites throughout the brain compared to those in the rat brain except for a few areas. By contrast, the rat exhibited high [3H]MK-801 binding sites in various brain regions, as compared with the gerbil brain. Thus, the gerbil differ from the rat with respect to the binding sites of major second messengers and neurotransmitters in the brain. The results may help better elucidate the relationship or species difference between gerbils and rats for neuronal function and behavioral pharmacology.