Introduction Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with airway hyperresponsiveness, which in turn is linked to abnormal Ca2+ homeostasis in airway smooth muscle (ASM). However, whether cigarette smoke (CS) directly alters or regulates Ca2+ signaling in human ASM cells (hASMC) is not fully known. In airway epithelial cells, acute CS exposure increases [Ca2+]i (Rasmussen et al, 2014). We speculate that CS disrupts Ca2+ homeostasis in hASMC, leading to exacerbated ASM contraction especially under inflamed conditions in COPD. Methods Human lung tissue was provided from the Royal Victoria Infirmary, Newcastle (ethical approval ref 11109/2016). Primary hASMC were isolated and maintained in culture up to 8 passages, and subjected to Ca2+ imaging studies using fura-2-AM. ASMC were exposed to either whole CS (1 cigarette) or CS extract (CSE; 1 cigarette bubbled in 25ml buffer solution) from 3R4F reference cigarettes (University of Kentucky) with the particulate phase filtered out. All data are presented as mean±SEM, and n numbers are independent experiments on cells from the same donor. Ca2+ response amplitudes are expressed as change in the fura-2 fluorescence ratio (F340/380), and rates expressed as change in F340/380 per minute (fitted using linear regression). Results We found that both CS and 10% CSE elevated [Ca2+]i in hASMC, which was abolished by removal of extracellular Ca2+, suggesting that both CS and CSE activate Ca2+ influx pathways in cultured hASMC (Fig. 1). Using a calcium-addback approach, we found that CSE-induced Ca2+ influx was poorly reversible after washing out CSE for 20 minutes (influx amplitude and rate following each addback before vs. after washout (n=7 for both sets) 0.19±0.04 vs. 0.16±0.04, p=0.16 and 0.20±0.04 vs. 0.14±0.04, p<0.05; Wilcoxon matched-pairs signed rank test). In addition, pre-treatment with cyclopiazonic acid to activate store-operated Ca2+ entry (SOCE) did not abolish CS- and CSE-induced Ca2+ influx. Furthermore, CSE-induced Ca2+ influx was not reduced by felodipine, an inhibitor of voltage-gated Ca2+ channels (VOCC; influx amplitude and rate DMSO (n=13) vs. felodipine-treated (n=16) 0.13±0.03 vs. 0.12±0.03, p=0.97 and 0.08±0.02 vs. 0.10±0.03, p=0.47; Mann-Whitney test), but was significantly attenuated by the lanthanide ion Gadolinium, Gd3+ (influx amplitude and rate untreated (n=11) vs. Gd3+-treated (n=6) 0.43±0.05 vs. 0.12±0.03, p<0.0005 and 0.43±0.07 vs. 0.10±0.03, p<0.0005; Mann-Whitney test). Conclusion Whole CS and CSE activate Ca2+ influx pathways in hASMC that are independent from SOCE and VOCC. The poorly reversible activation of Ca2+ influx may lead to long-term elevation of hASMC [Ca2+]i in chronic smokers, contributing to airway hyperresponsiveness. Further investigations into the identity of Ca2+ influx channels activated, and the direct effects of CS/CSE on ASM contractile mechanisms are on-going.