Recent results showing that large conductance, calcium-activated K⁺ channels (BK_Ca channels) undergo direct tyrosine phosphorylation in the presence of c-Src tyrosine kinase have suggested the involvement of these channels in Src-mediated signaling pathways. Given the important role for c-Src in integrin-mediated signal transduction, we have examined the potential regulation of BK_Ca channels by Pyk2, a calcium-sensitive tyrosine kinase activated upon integrin stimulation. Transient co-expression of murine BK_Ca channels with either wild-type Pyk2 or Hck, a Src-family kinase, led to an enhancement of BK_Ca channel activity over the range of 1-10 µM free calcium, whereas co-expression with catalytically inactive forms of either kinase did not significantly alter BK_Ca gating when compared to channels expressed alone. In the presence of either wild-type Pyk2 or Hck, BK_Ca subunits were found to undergo tyrosine phosphorylation, as determined by immunoprecipitation and Western blotting strategies. However, tyrosine phosphorylation of the BK_Ca subunit was not detected for channels expressed alone or together with inactive forms of either Pyk2 or Hck. Interestingly, wild-type, but not inactive, Pyk2 was also present in BK_Ca channel immunoprecipitates, suggesting that Pyk2 may co-associate with the BK_Ca channel complex following phosphorylation. Collectively, the observed modulation and phosphorylation of BK_Ca channels by Pyk2 and a Src-family kinase may reflect a general cellular mechanism by which GPCR and integrin activation lead to the regulation of membrane ion channels.