A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca(2+)-dependent Cl(-) channels via stimulation of Ca(2+) entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca(2+) 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. On the basis of these studies, the Ca(2+) entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X(4), P2X(5), and P2X(6) subtypes in non-CF (16HBE14o(-)) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca(2+) entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X(4) and P2X(6) (but not P2X(5)) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca(2+) entry markedly in fura-2 Ca(2+) measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca(2+) entry channel subtypes are expressed in airway epithelia. P2X(4) and P2X(6) may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype.