[Background] Recent studies of immune cells and their roles in maintaining homeostasis in the body, and protecting it from not only viral and bacterial infections but also tumors, have suggested that mast cell cytokines may play important roles. [Purpose] The present study was undertaken to investigate mast cell melatonin synthesis and its role in homeostasis. [Methods] mRNA expression in LAD2 cells, a human mast cell-derived cell line, was examined for aralkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), key enzymes for melatonin biosynthesis. [Results] mRNA for both enzymes was expressed in the LAD2 cells. Enzyme levels were enhanced by stimulation with ATPγ-S (100 μM) without β-hexosaminidase (β-Hex) release, while BzATP (100 μM) stimulation resulted in β-Hex release but no increase in mRNA. [Conclusion] These results suggest that melatonin release from mast cells is not involved in the allergic response, but is involved in homeostasis.