Large conductance calcium- and voltage- activated potassium (BK) channels assemble as macromolecular signalling complexes and are potently regulated by reversible protein phosphorylation. However, although numerous studies have revealed regulation of BK channels through changes in direct phosphorylation of the pore-forming -subunits the functional role of changes in phosphorylation of defined adapter/signalling proteins within the complex on channel function are essentially not known. Here, we demonstrate that mammalian BK channels are potently regulated by endogenous protein tyrosine kinase and protein tyrosine phosphatase activity closely associated with the channel. BK channel regulation was not dependent upon direct phosphorylation of the BK -subunit, rather channel function was controlled by the tyrosine phosphorylation status of the adapter protein cortactin that assembles directly with the BK channel. Our data thus reveals a novel mode for BK channel regulation by reversible tyrosine phosphorylation and strongly supports the hypothesis that phosphorylation dependent regulation of accessory proteins within the BK channel signalling complex represents an important target for control of BK channel function.