SKCa (small-conductance Ca(2+)-activated K(+)) channels are widely distributed in different tissues, including the brain, pancreatic islets and myocardium and play an important role in controlling electrical activity and cellular functions. However, intracellular signal modulation of SKCa channels is not fully understood. The present study was designed to investigate the potential regulation of hSKCa1 (human SKCa1) channels by PTKs (protein tyrosine kinases) in HEK (human embryonic kidney)-293 cells expressing the hSKCa1 (KCNN1) gene using approaches of whole-cell patch voltage-clamp, immunoprecipitation, Western blotting and mutagenesis. We found that the hSKCa1 current was inhibited by the broad-spectrum PTK inhibitor genistein, the selective EGFR (epidermal growth factor receptor) kinase inhibitors T25 (tyrphostin 25) and AG556 (tyrphostin AG 556), but not by the Src-family kinases inhibitor PP2. The inhibitory effect of these PTK inhibitors was significantly antagonized by the PTP (protein tyrosine phosphatase) inhibitor orthovanadate. The tyrosine phosphorylation level of hSKCa1 channels was reduced by genistein, T25 or AG556. The reduced tyrosine phosphorylation was countered by orthovanadate. Interestingly, the Y109F mutant hSKCa1 channel lost the inhibitory response to T25 or AG556, and showed a dramatic reduction in tyrosine phosphorylation levels and a reduced current density. These results demonstrate the novel information that hSKCa1 channels are inhibited by genistein, T25 and AG556 via EGFR tyrosine kinase inhibition, which is related to the phosphorylation of Tyr(109) in the N-terminus. This effect may affect electrical activity and cellular functions in brain, pancreatic islets and myocardium.