Large-conductance Ca(2+)-activated K(+ )(BK) channel regulates action potential firing in pancreatic β-cells. We cloned novel spliced variants of the BK-channel β(2)-subunit (BKβ2b), which consisted of 36 amino acids including the N-terminal in the original human BKβ2 (BKβ2a), from human and rodent pancreas. Real-time PCR analysis showed the abundant expression of BKβ2b transcripts in human and rodent pancreas and also in the RINm5f insulinoma cell line. In addition, up-regulation of both BK-channel α-subunit (BKα) and BKβ2b transcripts was observed in pancreas tissues from diabetes mellitus patients. In HEK293 cells co-expressing BKα and BKβ2b, the inactivation of BK-channel currents, which is typical for BKα + BKβ2a, was not observed, and electrophysiological and pharmacological properties of BKα + BKβ2b were almost identical to those of BKα alone. In HEK293 cells stably expressing BKα, the transient co-expression of yellow fluorescence protein (YFP)-tagged BKβ2a proteins resulted in their distribution along the cell membrane. In contrast, the co-expression of YFP-tagged BKβ2b with BKα showed diffusely distributed fluorescence signals throughout the cell body. Taken together, the predominant splicing of BKβ2b versus that of BKβ2a presumably enhances the contribution of BK channels to membrane potential and may possibly be a factor modulating insulin secretion in a suppressive manner in pancreatic β-cells.