Visualization of brain activity in humans and animals using functional magnetic resonance imaging (fMRI) is an established method for translational neuropsychopharmacology. It is useful to study the activity of defined brain structures, however it requires further refinement to allow more specific cellular analyses, like for instance, the activity of selected pools of brain cells. Here, we investigated brain activity in serotonergic pathways in the adult mouse brain by using acute pharmacological challenge of 5-hydroxytryptamine (5-HT) 1A receptors. We show that administration of the 5-HT(1A) receptor agonist 8-OH-DPAT prompts a dose-dependent reduction in local cerebral blood volume (CBV) in brain areas rich in neurons expressing post-synaptic 5-HT(1A) receptor, including the prefrontal cortex, hippocampus and amygdalar nuclei. Region-specific inhibition of the response by co-injection of 8-OH-DPAT with the selective 5-HT(1A) receptor antagonist WAY-100635, or in 5-HT(1A) knock-out mice, suggests that 5-HT(1A) receptors are the primary targets of the agonist. Overall, the data demonstrate the feasibility of mapping region-specific serotonergic transmission in the adult mouse brain in vivo by non-invasive fMRI. The method opens novel perspectives for investigating 5-HT(1A) receptor functions in mouse models of human pathologies resulting from a dysfunction of the 5-HT(1A) receptor or the serotonergic system, including depression and anxiety.