© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd. Although activated T lymphocytes express tryptophan hydroxylase 1 and produce 5-HT, the metabolic fate and cellular handling of this 5-HT is unclear. Here, we investigated key proteins in T cells linked to 5-HT metabolism and storage and compare differences in 5-HT synthesis and metabolism between T-cell subsets. Methods: We cultured human Jurkat T cells and mouse splenic CD3+, CD4+ and CD8+ T cells with or without T-cell activators (phorbol ester/ionomycin, concavalin A or plate-bound anti-CD3 antibody). Subsequently, we measured mRNA and/or protein for monoamine oxidase A and B, vesicular monoamine transporter 1 and 2, N-acetyl transferase and tryptophan hydroxylase 1. In addition, we measured the release of exogenously loaded [3H]5-HT and endogenously synthesized 5-HT from CD4+ and CD8+ T-cell subsets. Results: Human and mouse T cells selectively express monoamine oxidase A. Following T-cell activation, mRNA levels of MAO-A increase robustly in parallel with tryptophan hydroxylase 1. Concomitant with these changes, T cells increase the expression of the type 1 vesicular monoamine transporter. Raised intracellular [Ca2+] rapidly releases preloaded [3H]5-HT from CD4+ and CD8+ T cells indicating that these cells have the capacity for the storage and regulated secretion of 5-HT. Notably, both the expression of tryptophan hydroxylase 1 and monoamine oxidase A, and 5-HT production are significantly greater in CD8+ compared with CD4+ T cells. Conclusion: These data reveal coordinated changes in 5-HT production, metabolism and storage that may optimize 5-HT secretion from the CD8+ T cell subset in response to activation stimuli.