The serotonin receptor subtype 6, which raises intracellular cyclic AMP via stimulatory G-proteins, has recently been cloned and characterized. To determine the distribution of serotonin subtype 6 messenger RNA, in situ hybridization was performed in coronal sections of rat brain. 35S-labeled riboprobe, complementary to the 5' non-coding region of the serotonin subtype 6 messenger RNA, and a 33P-labeled riboprobe complementary to its 3' non-coding region, were used for hybridization. Serotonin subtype 6 receptor message was found in serotonin projection fields, rather than regions of serotonin-containing cell bodies, suggesting that the receptor is mainly postsynaptic. Hybridization signal was highest in olfactory tubercle, as well as prominent in the striatum, nucleus accumbens, dentate gyrus, and CA1, CA2 and CA3 of the hippocampus. Less intense hybridization was observed in cerebellum, some diencephalic nuclei, the amygdala, and layers 2, 3, 4 and 6 of the cortex. This pattern of hybridization was observed with both probes, but not when sense transcripts were used. Because the serotonin subtype 6 receptor has a high affinity for the atypical antipsychotic clozapine, and because striatum and nucleus accumbens are proposed sites of antipsychotic drug effects, the possibility is raised that this receptor may play an important role in mediating the effects of the atypical antipsychotic agents.