Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK₁R, NK₂R, and NK₃R) classically couple to Gq protein-mediated inositol 1,4,5-triphosphate (IP3) synthesis and liberation of intracellular calcium [Ca²⁺]_i which initiates contraction but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle and were subsequently overexpressed in human airway smooth muscle cells using an HIV-1-based lentivirus transduction system. Specific NKR agonists [NK₁R: [Sar⁹, Met(O₂)¹¹]-substance P, NK₂R: [-ala⁸]-NKA (4-10), NK₃R: senktide] stimulated inositol phosphate synthesis and increased [Ca²⁺]_i in native human airway smooth muscle cells and human airway smooth muscle cells transfected with each NKR subtype. These effects were blocked by NKR selective antagonists (NK₁R: L-732138, NK₂R: GR159897, NK₃R: SB222200). The initial transient and sustained phases of increased [Ca²⁺]_i were predominantly inhibited by the IP3 receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB), or the store-operated calcium channel antagonist, SKF96365, respectively. These results show that all 3 subtypes of NK receptors are expressed in native human airway smooth muscle cells and that IP3 levels are the primary mediators of NKRs-stimulated initial [Ca²⁺]_i increases, whereas store-operated Ca²⁺ channels mediate the sustained phase of the [Ca²⁺]_i increase.