The primary event in most types of pain is depolarization of peripheral nociceptive afferents. Ion channels control the excitability of nociceptors and Ca2+ activated Cl- channel ANO1 (TMEM16A) is one of the most recently discovered pro-algesic ion channels expressed in these neurons (1, 2). In order to investigate activation of ANO1 in nociceptors by intracellular Ca2+ signals we developed a single-cell imaging approach that allows simultaneous monitoring of Cl- channel activity and intracellular Ca2+ concentration in cultured dorsal root ganglion (DRG) neurons. To this end we transfected DRG cultures with the halide-sensitive H148Q/I152L EYFP mutant (3). ANO1 is highly permeable to I- ions (4) and H148Q/I152L EYFP fluorescence is quenched by I-. Since excitation/emission maxima for YFP are within visible light spectrum, it is possible to combine YFP imaging with the conventional fura-2 Ca2+ imaging. Equimolar substitution of NaCl by NaI in the extracellular solution resulted in prominent run-down of YFP fluorescence, likely due to the background anionic permeability. Therefore, we first optimised the extracellular I- concentration using GABA to activate GABAA receptors that are abundantly expressed in DRG neurons. Extracellular solutions containing 5-30mM NaI were used and the speed of the YFP fluorescence rundown and fluorescence response to GABA were analyzed. The GABA responses were similar within the tested NaI concentration range, however, 5mM NaI gave the lowest rundown (1.1±0.01% over 50s, n=8 as compared to 1.6±0.01% and 15.5±0.05% for 10mM and 30 mM NaI respectively). We then investigated activation of Ca2+-activated anion conductance in DRG neurons in response to i) Ca2+ release from the IP3-sensitive intracellular stores and ii) Ca2+ influx via the voltage-gated Ca2+ channels. The former was induced by the Gq/11-coupled receptor agonist bradykinin (BK) and the latter by depolarizing neurons with extracellular solution containing 50mM KCl. BK induced a significantly greater reduction of YFP fluorescence as compared to that produced by depolarization; 47±4% (n=15) vs 30±3% (n=27) respectively. Global intracellular Ca2+ increases induced by BK and depolarization were of similar amplitude. The decreases in YFP fluorescence induced by BK and depolarization were inhibited by the Cl- channel blocker niflumic acid. We also performed dual YFP and Ca2+ imaging to compare BK response to that produced by TRPV1 agonist capsaicin (CAP). Again, BK produced significantly larger quenching of YFP fluorescence as compared to that of CAP. Moreover, the onset of the YFP response to CAP had ~50s delay relative to the onset of the Ca2+ transient; no such delay was seen with BK. CAP induced larger Ca2+ transients as compared to BK. Our results suggest that ANO1 in nociceptive DRG neurons is preferentially coupled to Ca2+ release from the ER.