Introduction: Prostate cancer is the second most common cancer in males worldwide and is the leading cause of death in males in the Western world. Our preliminary screen of prostate cancer tumour tissues indicated that large conductance, Ca2+-activated K+ (BK) channels could be implicated in prostate cancer biology. The aim of the present study was therefore to investigate the role of BK channels in prostate cancer and normal prostate cell lines. Materials and Methods: A panel of cell lines including prostate cancer cells (PC3, DU145) and immortalised normal prostate cells (PNT2) were used. BK functional expression was investigated with channel inhibitors in clonogenic cell survival assays, scratch-wound healing assays and immunofluorescence. Quantitative data are expressed as mean ± SEM. The unpaired Student’s t-test was used for statistical comparisons) with P<0.05 considered to be significant.Results: Prostate cancer DU145 and PC3 cells and normal prostate PNT2 cells were labelled with antibodies to the BK channel alpha-subunit, KNCMA1 and imaged with confocal microscopy. The BK channel was expressed in all 3 cell lines with expression in the cytoplasm and perinuclear region. The potential role of BK channels on cell survival was examined using the inhibitors tetrathylammonium chloride (TEA) and paxilline1 in clonogenic survival assays. TEA (0.05 – 5mM, N=3) caused a concentration-dependent reduction in cell survival in DU145 (P<0.001, 5mM) and PC3 cells (P<0.01, 1mM; P<0.001, 5mM). In contrast, TEA increased the survival of normal PNT2 cells (N=2) with increased survival fractions at 0.05, 0.1, 0.5 and 5mM, P<0.05). Paxilline (5nM – 10µM) had little effect on cell survival in prostate cancer DU145 (N=3) or PC3 (N=3) cells but increased survival of normal PNT2 cells (P<0.05). In scratch-wound healing assays indicative of migration, TEA (5mM) did not significantly affect % wound closure in DU145 or PC3 cells compared with controls (N=3, P>0.05). Paxilline (1 µM) significantly increased wound closure by 34% in DU145 cells (P<0.01, N=2) but had little effect on wound closure (P>0.05, N=2) in PC3 cells. Conclusions: BK/KCNMA1 channels are functionally expressed in the normal prostate epithelial and prostate cancer cells examined. Inhibition of BK channels with TEA or paxilline reduced survival of prostate cancer cells but had less effect on migration. In contrast, BK inhibition enhanced the survival of normal prostate PNT2 cells. These results indicate that BK channels underpin different cellular processes in normal vs malignant prostate epithelial cells.