Calcium-activated chloride channels (CaCC) are major targets for an alternative anion channel therapy in cystic fibrosis (CF). The molecular identity of CaCC has yet to be defined. The bestrophins are a novel candidate for the molecular origin of CaCC (Sun et al. 2001; Qu et al. 2004). In this study, we provide the first evidence for the existence of human bestrophin 1 (hBest 1) in CFPAC-1 cells. Native CaCC were characterised in confluent monolayers of CFPAC-1 cells using a non-radioisotope, iodide efflux assay. Application of ionomycin to CFPAC-1 monolayers led to a concentration-dependent increase in iodide efflux. Maximal efflux was observed with 2 μM ionomycin, which induced an increase in efflux of 14.6 ± 1.9% (n = 4, p<0.001). Niflumic acid (200 μM) inhibited the response demonstrated with 0.5 μM ionomycin from 7.7 ± 1.3% to 1.8 ± 1.3% (n = 4, p<0.001). The purine receptor agonist, uridine 5’-triphosphate (UTP), also stimulated efflux from CFPAC-1 monolayers in a concentration dependent manner. Maximal efflux was observed at 200 μM UTP (9.0 ± 1.6%, n = 6, p<0.001). Niflumic acid (200 μM) reduced the response elicited by 100 μM UTP from 7.9 ± 2.0% to 1.99 ± 0.13% (n = 6, p<0.001) and 500 μM 4,4’-diisothiocyanatostilbene-2,2’-disulfonate (DIDS) also reduced the response from 7.9 ± 2.0% to 2.2 ± 1.5% (n = 6, p<0.001). Total RNA was isolated from CFPAC-1 cells and RT-PCR was carried out with hBest1 specific primers corresponding to three areas of the cDNA sequence (NM 004183, Genbank database). RT-PCR yielded three bands of 489, 461 and 411 base pairs, respectively, identical in size to the predicted products. Sequence analysis of the three products demonstrated 100% sequence identity to that published in GenBank, indicating that the three products were consistent with the hBest1 gene. To assess the expression of hBest 1 at the cellular level, two commercially available antibodies were used, ab 14927 (Abcam, UK) and Bst 121 (Fabgennix, USA). Western blot analysis of CFPAC-1 protein isolates showed that both antibodies gave similar blots (n=8 and n=4, respectively), with a principal band at approximately 55 kDa and three very faint bands around 70, 75 and 78 kDa. To further confirm the expression of hBest 1 in CFPAC-1 cells, the cells were fixed with 4% paraformaldehyde and hBest1 was labelled using either ab 14927 (n=3) or Bst 121 (n=2). Preliminary confocal image analysis of the fixed cells showed that hBest 1 is expressed to a large degree in the cytoplasm, most probably in cytoplasmic vesicles. Although not immediately obvious, some staining may be confined to the plasma membrane. Taken together, these preliminary data suggest that hBest 1 is expressed in the CFPAC-1 cell line. Further studies are being undertaken to determine whether other bestrophin homologues are expressed in CFPAC-1 cells.