Serotonin is known to be present in pancreatic beta-cells and to play several physiological roles, including insulin secretion, beta-cell proliferation, and paracrine inhibition of cc-cells. However, the serotonin production of different cell lines and islets has not been compared based on age, sex, and diabetes related conditions. Here, we directly compared the serotonin concentrations in beta TC and MIN6 cell lines, as well as in islets from mice using ultra-performance liquid chromatography tandem mass spectrometry. The average serotonin concentration was 5-10 ng/mg protein in the islets of male and non-pregnant female mice. The serotonin level was higher in females than males at 8 weeks, although there was no difference at 1 year. Furthermore, we observed serotonin by immunofluorescence staining in the pancreatic tissues of mice and human. Serotonin was detected by immunofluorescence staining in a portion of beta-cells from islets of old female mice, but not of male or young female mice. A similar pattern was observed in human pancreas as well. In humans, serotonin production in beta-cells was associated with a diabetes-free condition. Thus, serotonin production in beta-cells was associated with old age, female sex, and diabetes-free condition. (C) 2017 Elsevier Inc. All rights reserved.