The BK (large conductance voltage and Ca2+ activated K+) channel is a key regulator of bladder smooth muscle contractility. To our knowledge in bladder smooth muscle the BK channel pore forming α subunit BKα associates in homotetramers with 4 regulatory smooth muscle specific β1 subunits. We challenged this concept in identify whether other regulatory BKβ subunits exist in mouse and rat bladder smooth muscle.We used a novel approach with single cell reverse transcriptase-polymerase chain reaction combined with immunocytochemical studies in freshly isolated mouse and rat bladder smooth muscle cells. Western blot was also performed.Reverse transcriptase-polymerase chain reaction identified the mRNA expression of various BK channel subunits in freshly isolated bladder smooth muscle cells. Our data indicate that, in addition to BKα and BKβ1, neuronal specific BKβ4 is expressed in mouse and rat bladder smooth muscle cells. BKβ4 expression was also revealed by Western blot. Immunocytochemistry was further applied to confirm the specific expression of BKβ4 protein directly in freshly isolated mouse and rat bladder smooth muscle cells.To our knowledge we performed the first comprehensive examination of the expression of BKα and BKβ subunits in bladder smooth muscle. We identified that the bladder smooth muscle BK channel has a distinctive architecture involving pore forming BKα and regulatory BKβ1/β4. Further studies of the functional roles of BKα, BKβ1 and BKβ4 directly in human bladder smooth muscle may help the development of alternative therapeutic strategies to control bladder dysfunction. New drugs targeting specific BK channel subunits in human bladder smooth muscle may prove useful for overactive bladder.