Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca2+]i in human CF (IB3-1) and non-CF (16HBE14o-) airway epithelial cells. Cytosolic Ca2+ was measured by fluorospectrometry using the fluorescent dye Fura-2/ AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca2+]i derived from intracellular stores in a Na+-rich environment. In contrast, ATP induced an increase in [Ca2+]i that had transient and sustained components in a Na+-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca2+]i only in Na+-free medium, suggesting competition between Na+ and Ca2+ through P2XRs. Biochemical evidence showed that the P2X4 receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca2+ channels, voltage-dependent Ca2+ channels, or Na+/Ca2+ exchanger in the ATP-induced sustained Ca2+ signal was ruled out. In conclusion, these data show that epithelial P2X4 receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca2+]i. In airway epithelia, a P2XR-mediated Ca2+ signal may have therapeutic benefit for CF.