We have shown that N-methyl-D-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux, and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca2+ and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells we assessed TNF (10ng/mL, 48h) effect on NMDA-R subunit abundance by quantitative PCR (qPCR), confocal imaging and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression, and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca2+ flux in Fura-2 loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca2+ mobilization and changed the temporal pattern of Ca2+ flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin cut lung slices (TCLS) from allergen-naïve animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from HDM-challenged mice and in allergen-naïve TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by selective NMDA-R antagonist, D-AP5, and bronchodilator responses were prevented by L-NAME (NOS inhibitor) or indomethacin (COX inhibitor). Collectively, we show that TNF augments NMDA-R mediated Ca2+ mobilization in HASM cells,while in multicellular TCLSs, allergic inflammation and TNF exposure leads to NMDA-R mediated bronchodilation. These findings reveal unique contribution of ionotrophic NMDA-R to airway hyperreactivity.