Stress and anxiety may worsen atopic dermatitis (AD) through the serotonin system. Serotonergic expression was measured in 28 patients with AD in relation to extent of the disease (SCORing of Atopic Dermatitis; SCORAD), pruritus intensity (visual analogue scale; VAS), anxiety traits (Swedish Universities Scales of Personality; SSP) and depression (Montgomery-Åsberg Depression Rating Scale-Self assessment; MADRS-S). Biopsies were taken from lesional and non-lesional AD skin, and investigated for expression of serotonin, its receptors 5-HT1A and 5-HT2, and serotonin transporter protein (SERT), using immunohistochemistry. 5-HT1AR-immunoreactivity (ir) was higher in lesional skin in apical epidermis and in mast cell-like cells in dermis, and 5-HT2AR-ir in apical epidermis and on blood vessels. In contrast, a basement membrane 5-HT2AR-ir signal was higher in non-lesional skin. The distribution of SERT-ir in the basal epidermal layer was higher in lesional skin. Positive and negative correlations were found between serotonergic markers and SCORAD, inflammation, pruritus intensity, anxiety traits, and depression score, indicating that serotonergic mechanisms are involved in AD.