Rationale: Substance P (SP) and hemokinin-1 (HK-1) are neuropeptides (NPs) that promote inflammatory responses by signaling through the neurokinin-1 receptor (NK-1R). Antagonists of NK-1R are highly effective in allergic inflammation and airway hyperresponsiveness in mice but lack efficacy in humans. The reason for this difference is unknown. Human mast cells express both the NK-1R and Mas-related G protein receptor X2 (MRGPRX2) which are both activated by SP. The objective of this study was to determine if HK-1 activates human mast cells via MRGPRX2. Moreover, since MRGPRX2 contains a cholesterol recognition amino acid consensus (CRAC) domain the interaction between MRGPRX2 and cholesterol in lipid rafts likely contributes to MRGPRX2 function. Another objective in this study is to investigate whether lipid rafts are associated with MRGPRX2 function. Materials and Methods: Flow cytometry was used to determine the expression of NK-1R and MRGPRX2 in a human mast cell line (LAD2). MRGPRX2 function was investigated by using SP and HK-1 to induce degranulation with a selective NK-1R antagonist (CP96345). Methyl-b-cyclodextrin (MbCD) was used for cholesterol depletion in this study. In addition, mutants of the CRAC domain on MRGPRX2 were used to compare the functions of the receptor on degranulation and Ca2+ mobilization in response to SP, HK-1, and other compounds (ciprofloxacin, HOE 140 and compound 48/80). Confocal microscopy was used to determine the localization of lipid raft compartments and MRGPRX2. Results: LAD2 cells expressed both MRGPRX2 and NK-1R. HK-1 and SP induced degranulation in LAD2 cells and RBL-2H3 cells stably expressing MRGPRX2, but this response was resistant to inhibition by an NK-1R inhibitor (CP96345). However, SP and HK-1 induced degranulation in RBL cells transiently expressing NK-1R and this response was inhibited by CP96345. Depleting cholesterol by using MbCD decreased the degranulation response. Furthermore, confocal microscopy showed that the lipid rafts and MRGPRX2 colocalized in both WT and the CRAC domain mutants. However, CRAC domain mutants of MRGPRX2 did not respond to SP in Ca2+ mobilization and degranulation assays. Discussion: This study provides a potential explanation for the previous observation that NK-1R antagonist are highly effective in allergic responses in mice but fails in human. Our findings suggest that unlike the situation in mice where the effects of neuropeptides are mediated via NK-1R, these effects are mediated by MRGPRX2 in humans. Moreover, lipid rafts may not be associated with MRGPRX2 function. However, the CRAC domain mutant appears to be defective in coupling G protein. Conclusion: This finding suggests that MRGPRX2 may serve as a novel target for modulating asthma and other neuropeptide/mast cell-mediated diseases.