The cysteinyl leukotrienes (LTs), LTC₄, LTD₄, and LTE₄, are potent inflammatory mediators and are involved in allergic reactions, such as bronchoconstriction, eosinophilic inflammation, and allergic cell proliferation. The present study aimed to elucidate the role of constitutively produced cysteinyl LTs in mast cell activation. We used a newly developed quantification method based on mass spectrometry to detect cysteinyl LTs in the cultured medium of mouse bone marrow-derived mast cells (BMMCs), which were obtained by interleukin (IL)-3-conditioned culture of mouse bone marrow. BMMCs were stimulated with immunoglobulin (Ig) E and antigen (IgE/Ag) or lipopolysaccharide for 1 or 24 h. This new quantification method revealed that unstimulated BMMCs produced and secreted LTB4 and LTE4 after 24 h of incubation. The treatment of unstimulated BMMCs for 2 h with montelukast, an antagonist of a cysteinyl LT receptor, CysLT1, resulted in the suppression of a downstream signaling event of this receptor, i.e., the decrease in phosphorylation of extracellular responsive kinases. Thus, cysteinyl LTs constitutively simulate BMMCs through the CysLT1 receptor in an autocrine manner. Treatment of BMMCs for 3 weeks with montelukast, which caused long-term inhibition of the autocrine cyteinyl LT-derived signal, significantly attenuated the IgE/Ag-dependent degranulation, as judged by the decrease in the release of β-hexosaminidase, an enzyme contained in the granules, whereas the production of cytokines, such as IL-6 and tumor necrosis factor-α, were largely unaffected. In conclusion, an autocrine signal derived from constitutively produced cysteinyl LTs predisposes mast cells to the degranulation upon allergic stimulation.