IMPORTANCE The cerebral serotonin 4 (5-HT4) receptor is a promising novel target for treatment of major depressive disorder (MDD), and pharmacological stimulation of the 5-HT4 receptor has been associated with improved learning and memory in healthy individuals.OBJECTIVE To map the neurobiological signatures of patients with untreated MDD compared with healthy controls and to examine the association between cerebral 5-HT4 receptor binding and cognitive functions in the depressed state.DESIGN, SETTING, AND PARTICIPANTS This case-control study used baseline data from the NeuroPharm clinical depression trial in Denmark. Adult participants included antidepressant-free outpatients with a current moderate to severe depressive episode and healthy controls. All participants completed positron emission tomography (PET) scanning with [C-11]SB207145 for quantification of brain 5-HT4 receptor binding, but only the patients underwent cognitive testing. Data analyses were performed from January 21, 2020, to April 22, 2022.MAIN OUTCOMES AND MEASURES The main study outcome was the group difference in cerebral 5-HT4 receptor binding between patients with MDD and healthy controls. In addition, the association between 5-HT4 receptor binding and verbal memory performance in the patient group was tested. Other cognitive domains (working memory, reaction time, emotion recognition bias, and negative social emotions) were assessed as secondary outcomes.RESULTS A total of 90 patients with untreated MDD (mean [SD] age, 27.1 [8.2] years; 64 women [71.1%]) and 91 healthy controls (mean [SD] age, 27.1 [8.0] years; 55 women [60.4%]) were included in the analysis. Patients with current MDD had significantly lower cerebral 5-HT4 receptor binding than healthy controls (-7.0%; 95% CI, -11.2 to -2.7; P = .002). In patients with MDD, there was a correlation between cerebral 5-HT4 receptor binding and verbal memory (r = 0.29; P = .02).CONCLUSIONS AND RELEVANCE Results of this study show that cerebral 5-HT(4 )receptor binding was lower in patients with MDD than in healthy controls and that the memory dysfunction in patients with MDD was associated with lower cerebral 5-HT4 receptor binding. The cerebral 5-HT4 receptor is a promising treatment target for memory dysfunction in patients with MDD.