Interstitial cells (ICs), analogous to the ICs of Cajal in the gut, may generate phasic activity (PA) in smooth muscle tissues including the bladder (1). Established markers of ICs include c-kit and vimentin. However, recent studies have shown that Anoctamin-1 (Ano1), a Ca2+-activated chloride channel, influences generation of pacemaker activity in gut ICs and may be used as a novel marker for these cells (2).Thus, our aim was to investigate the expression Ano1 in ICs of the rat bladder and to explore the role of T16Ainh-A01, a novel specific inhibitor of Ano1 (3), in modulating the phasic activity (PA) of the bladder tissue.Wistar rats (p19-23) were used. For immunolabelling studies, whole bladders (N=3), were stretched and fixed, and bladder sheets were cut into strips, washed and blocked with 1% horse serum in PBS+0.5% Triton. Tissues were incubated with primary antibodies (Ano1 1:250, vimentin 1:500, overnight, 4°C) washed, incubated with secondary antibodies (1h, room temperature) and DAPI nuclear stain, mounted on slides and examined using Leica DMI6000 inverted microscope. Longitudinal strips (5-8mm) of intact bladder were mounted in perspex microbaths, superfused with Krebs’ solution and maintained at 37°C. Isometric tension was measured via UF1 force transducers connected to a Powerlab using LabChart software. After 1h of equilibration, the effect of T16Ainh-A01 (Tocris) (3 or 10µM, 30min exposure for each concentration) or drug vehicle (DMSO) on PA was investigated by measuring amplitude and frequency of PA. Statistical analysis was carried out using Student’s paired t-test. Percentage change in the amplitude/frequency of PA was calculated in presence of T16Ainh-A01 relative to that in the absence of drug. Data is expressed as the mean±SEM. Ano1 staining was detected in a subpopulation of vimentin positive ICs in the mucosa and detrusor of rat bladder. 3μM (n=17) and 10μM (n=11) T16Ainh-A01 did not alter the amplitude of basal PA. Only 10μM T16Ainh-A01 demonstrated a slight, but insignificant decrease in frequency of PA (6.3±4.0%, p=0.1727).We have shown that Ano1 is expressed in a subpopulation of vimentin positive ICs in mucosa and detrusor layers of rat bladder. Commercially available T16Ainh-A01 did not have a significant effect on PA of rat bladder strips. Although 10μM dose demonstrated a slight and insignificant inhibition of frequency, it formed a precipitate in the buffer during drug exposure. Previous reports using T16Ainh-A01 tested isolated cells directly (4) and not whole tissues. Thus, lack of visible effects on bladder PA might be due to poor tissue penetration by the drug, lack of access to target cells or problems with dissolving of the compound. This study confirms Ano1 as a novel IC marker; however, functional role of this channel in rat bladder needs further investigation.