Neither Pseudomonas aeruginosa nor flagellin affected cytosolic Ca²⁺ concentration ([Ca]_i) in airway epithelial cell lines JME and Calu-3, but bacteria or flagellin activated NF-B, IL-8 promoter, and IL-8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca²⁺ pump, releases endoplasmic reticulum Ca²⁺, and triggers Ca²⁺ entry through plasma membrane channels) both increased [Ca]_i but hardly stimulated NF-B and IL-8. ATP and thapsigargin elicited larger, synergistic activations of NF-B and IL-8 secretion when combined with flagellin. BAPTA-AM (to buffer [Ca]_i) or Ca²⁺-free solution reduced increases in [Ca]_i due to ATP or thapsigargin and also reduced NF-B activation and IL-8 secretion triggered by flagellin, ATP, thapsigargin, ATP + flagellin, and thapsigargin + flagellin. IL-8 promoter analysis showed that AP-1 and CCAAT/enhancer-binding protein (C/EBP)/nuclear factor for IL-6 (NF-IL6) sites were important for IL-8 expression, and the NF-B-binding site was critical for activation by all agonists and for activation by [Ca]_i. Thus increased [Ca]_i was not required for P. aeruginosa- or flagellin-activated NF-B and IL-8 expression and secretion, and increased [Ca]_i was only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in [Ca]_i synergized with flagellin or P. aeruginosa, and buffering or reducing [Ca]_i reduced these responses. Thus [Ca]_i plays an important regulatory role in P. aeruginosa- or flagellin-activated innate immune responses in airway epithelia. Dose-dependent responses indicated that flagellin-ATP synergism occurred most prominently at ATP concentrations ([ATP]) > 10 µM and [flagellin] >10^–8 g/ml and during steady increases rather than oscillations in [Ca]_i.