A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca²⁺-dependent Cl^- channels via stimulation of Ca²⁺ entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca²⁺ in human airway epithelial cells that translates into stimulation of sustained secretory Cl^- transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. Based on these studies, the Ca²⁺ entry channels most likely involved were P2X purinergic receptor channels. In this study, molecular and biochemical data show co-expression of P2X₄, P2X₅, and P2X₆ subtypes in non-CF (16HBE14o^-) and CF (IB3-1) human bronchial epithelial cells. Other P2XR subtypes are rarely or not expressed in airway epithelia, epithelial cell models from other CF-relevant tissues, or in vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA (siRNA) fragments specific to P2X₄ and P2X₆ (but not P2X₅) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc and ATP-induced Ca²⁺ entry markedly in Fura-2 Ca²⁺ measurements and knocked down protein by more than 65%. These data suggest that multiple P2X receptor Ca²⁺ entry channel subtypes are expressed in airway epithelia. P2X₄ and P2X₆ may co-assemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype.