Endogenous serotonin (5-HT) release can be measured noninvasively using positron emission tomography (PET) imaging in combination with certain serotonergic radiotracers. This allows us to investigate effects of pharmacological and nonpharmacological interventions on brain 5-HT levels in living humans. Here, we study the neural responses to a visual stimulus using simultaneous PET/MRI. In a cross-over design, 11 healthy individuals were PET/MRI scanned with the 5-HT(1B)receptor radioligand [C-11]AZ10419369, which is sensitive to changes in endogenous 5-HT. During the last part of the scan, participants either viewed autobiographical images with positive valence (n= 11) or kept their eyes closed (n= 7). The visual stimuli increased cerebral blood flow (CBF) in the occipital cortex, as measured with pseudo-continuous arterial spin labeling. Simultaneously, we found decreased 5-HT(1B)receptor binding in the occipital cortex (-3.6 +/- 3.6%), indicating synaptic 5-HT release. Using a linear regression model, we found that the change in 5-HT1B receptor binding was significantly negatively associated with change in CBF in the occipital cortex (p= .004). For the first time, we here demonstrate how cerebral 5-HT levels change in response to nonpharmacological stimuli in humans, as measured with PET. Our findings more directly support a link between 5-HT signaling and visual processing and/or visual attention.