1. This study characterizes the K(+) channel(s) underlying charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. 2. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. 3. Single-channel, outside-out patch recordings revealed a 17.1+/-0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca(2+) demonstrated that the open probability was increased by Ca(2+). This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. 4. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1+/-0.7 mV) were reduced by apamin (100 nM; 17.0+/-1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 microM, 21.0+/-0.3 mV) were unaffected (21.7+/-0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. 5 A porcine homologue of the intermediate-conductance Ca(2+)-activated K(+) channel (IK1) was identified in endothelial cells. 6. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca(2+)-activated K(+) channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin-sensitive component of the EDHF response.