Large conductance calcium- and voltage- activated potassium (BK) channels are widely expressed in the mammalian central nervous system. Although the activity of BK channels in endocrine and vascular cells is regulated by protein kinases and phosphatases associated with the channel complex, direct evidence for such modulation in neurons is largely lacking. Single channel analysis from inside-out patches isolated from the soma of dissociated rat cerebellar Purkinje neurons demonstrated that the activity of BK channels is regulated by multiple endogenous protein kinases and protein phosphatases in the membrane patch. The majority of BK channels were non-inactivating and displayed a 'low' activity phenotype determined at + 40 mV and 1 mM intracellular free calcium. These channels were activated by cAMP-dependent protein kinase (PKA) associated with the patch and the extent of PKA- activation was limited by an opposing endogenous type 2A- like protein phosphatase (PP2A). Importantly, PKA activation was dependent upon the prior phosphorylation status of the BK channel complex dynamically controlled by protein kinase C (PKC) and protein phosphatase 1 (PP1). In contrast, Purkinje cells also displayed a low proportion of non-inactivating BK channels with a 'high' activity under the same recording conditions and these channels were inhibited by endogenous PKA. Our data suggest that: i) multiple endogenous protein kinases and phosphatases functionally couple to the BK channel complex to allow conditional modulation of BK channel activity in neurons and, ii) native, phenotypically distinct, neuronal BK channels are differentially sensitive to PKA-dependent phosphorylation.