It has been demonstrated that an unidentified cytosolic factor(s) reduces KACh channel function. Therefore, this study attempted to elucidate the cytosolic factor. Fresh cytosol isolated from normal heart (FC) depressed the KACh channel activity, but cytosol isolated from the ischemic hearts (IC) did not modulate the channel function. Electrophorectic analysis revealed that a protein of ∼80 kDa was markedly reduced or even lost in IC. By using peptide sequencing analysis and Western blot, this 80 kDa protein was identified as transferrin (receptor-mediated Fe3⁢ transporter, 76 kDa). Direct application of transferrin (100 nM) to the cytoplasmic side of inside-out patches decreased the open probability (Po, 12.7⁑6.4%, n=4) without change in mean open time (τo, 98.5⁑1.3%, n=4). However, the equimolar apotransferrin, which is free of Fe3⁢, had no effect on the channel activity (N*Po, 129.1⁑13.5%, n=3). Directly applied Fe3⁢ (100 nM) showed results similar to those of transferrin (N*Po: 21.1⁑3.9%, n=5). However Fe2⁢ failed to reduce the channel function (N*Po, 106.3⁑26.8%, n=5). Interestingly, trivalent cation La3⁢ inhibited N*Po of the channel (6.1⁑3.0%, n=3). Taken together, these results suggest that Fe3⁢ bound to transferrin can modulate the KACh channel function by its electrical property as a polyvalent cation.