Bovine pancreatic trypsin inhibitor (BPTI) is a 58 residue protein whose binding to various serine proteases has been extensively studied by X-ray crystallography. We have found that BPTI also binds to an intracellular site associated with the large conductance Ca(2+)-activated K+ channel, as detected by the production of subconductance events in single channels incorporated into planar lipid bilayers. BPTI is highly homologous to a family of mamba snake dendrotoxin proteins that inhibit various K+ channels at an extracellular site. BPTI thus provides a useful model system to explore basic mechanisms underlying protein-channel interactions.