Carbon monoxide (CO) is an important gas produced endogenously in humans by heme oxygenase (HO) enzymes (HO-1 and HO-2). CO is known to function as an anti-inflammatory agent, but its effects on airway inflammation and ion transport remain unclear. The effects of CO on cell damage- and nucleotide-induced pro-inflammatory cytokine release were investigated in cultured primary human bronchial epithelial (HBE) cells (ScienCell Research Laboratories, San Diego, CA, USA) and the 16HBE14o- HBE cell line. Its effects on calcium- and cAMP-dependent chloride (Cl−) secretion were examined using a technique that allowed the simultaneous measurement and quantification of real-time changes in signalling molecules (cAMP and Ca2+ levels) and ion transport in a polarised epithelium (1, 2). Changes in Fura-2 ratios and short circuit current (ISC; an index of electrogenic ion transport) were measured concurrently in polarised epithelial cells mounted in a miniature Ussing chamber. Real-time changes in cAMP levels were detected using an EPAC-based fluorescence resonance energy transfer (FRET) sensor. In this study, using RT-PCR and western blot analysis, we demonstrated that both HO-1 and HO-2 mRNAs and proteins were expressed in primary HBE and 16HBE14o- cells, respectively (n = 3-4). The upregulation of HO-1 protein expression by hemin (10 μM), a well-known HO-1 inducer, inhibited the cationic protein poly-L-arginine (1 μM; PLA)- or ATPγS (10 μM)-induced IL-6 and IL-8 release in primary HBE and 16HBE14o- cells, as quantified by ELISA (n = 5-10). CORM-3 (100 μM; a CO-releasing molecule) inhibited the ATPγS-induced polarised secretion of IL-6 and IL-8 in 16HBE14o- cells (n = 5-7). Furthermore, CORM-3 (100 μM) inhibited UTP (100 μM)-induced increases in calcium (Control: change in the Fura-2 ratio = 0.052 ± 0.007; +CORM-3: change in the Fura-2 ratio = 0.032 ± 0.003; p < 0.05; values are means ± S.E.M., compared using ANOVA) and ISC (Control: change in ISC = 26.79 ± 1.73 μAcm−2; +CORM-3: change in ISC = 12.45 ± 2.14 μAcm−2; p < 0.05; n = 8). CORM-3 (100 μM) also inhibited the forskolin (10 μM)-induced increase in the FRET ratio (Control: change in the ratio = 0.09 ± 0.01; +CORM-3: change in the ratio = 0.05 ± 0.01; p < 0.05) and subsequent increase in ISC (Control: change in ISC = 47.35 ± 2.80 μAcm−2; +CORM-3: change in ISC = 26.56 ± 2.59 μAcm−2; p < 0.05, n = 4-7). Overall, our data suggest that CO modulates the pro-inflammatory state of the human airway epithelia by interfering with the P2Y receptor-mediated signalling pathway. CO also modulates the Ca2+- and cAMP-dependent secretion of Cl− in the human airway epithelia. Thus, therapeutic strategies using HO-1-inducing agents or CORMs may be developed for treating airway-related inflammatory disorders, such as asthma.