Neither P. aeruginosa nor flagellin affected cytosolic [Ca²⁺], Ca_i, in airway epithelial cell lines JME or Calu-3, but bacteria or flagellin activated NF-B, IL8 promoter and IL8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca²⁺ pump, releases endoplasmic reticulum Ca²⁺ and triggers Ca²⁺ entry through plasma membrane channels) both increased Cai but hardly stimulated NF-B and IL8. ATP and thapsigargin elicited larger, synergistic activations of NF-B and IL8 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 IL8 secretion triggered by flagellin, ATP, thapsigargin, ATP+flagellin and thapsigargin+flagellin. IL8 promoter analysis showed that AP-1 and CEBP/NF-IL6 sites were important for IL8 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 IL8 expression and secretion, and increased Ca_i was only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in Cai 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] > 10 µM and [flagellin] >10^-8 g/ml and during steady increases rather than oscillations in Ca_i.