An inhibitor of the high conductance, Ca2(+)-activated K+ channel (PK,Ca) has been purified to homogeneity from venom of the scorpion Buthus tamulus by a combination of ion exchange and reversed-phase chromatography. This peptide, which has been named iberiotoxin (IbTX), is one of two minor components of the crude venom which blocks PK,Ca. IbTX consists of a single 4.3-kDa polypeptide chain, as determined by polyacrylamide gel electrophoresis, analysis of amino acid composition, and Edman degradation. Its complete amino acid sequence has been defined. IbTX displays 68% sequence homology with charybdotoxin (ChTX), another scorpion-derived peptidyl inhibitor of PK,Ca, and, like this latter toxin, its amino terminus contains a pyroglutamic acid residue. However, IbTX possesses 4 more acidic and 1 less basic amino acid residue than does ChTX, making this toxin much less positively charged than the other peptide. In single channel recordings, IbTX reversibly blocks PK,Ca in excised membrane patches from bovine aortic smooth muscle. It acts exclusively at the outer face of the channel and functions with an IC50 of about 250 pM. Block of channel activity appears distinct from that of ChTX since IbTX decreases both the probability of channel opening as well as the channel mean open time. IbTX is a selective inhibitor of PK,Ca; it does not block other types of voltage-dependent ion channels, especially other types of K+ channels that are sensitive to inhibition by ChTX. IbTX is a partial inhibitor of 125I-ChTX binding in bovine aortic sarcolemmal membrane vesicles (Ki = 250 pM). The maximal extent of inhibition that occurs is modulated by K+, decreasing as K+ concentration is raised, but K+ does not affect the absolute inhibitory potency of IbTX. A Scatchard analysis indicates that IbTX functions as a noncompetitive inhibitor of ChTX binding. Taken together, these data suggest that IbTX interacts at a distinct site on the channel and modulates ChTX binding by an allosteric mechanism. Therefore, IbTX defines a new class of peptidyl inhibitor of PK,Ca with unique properties that make it useful for investigating the characteristics of this channel in target tissues.