Conus snail (Conus) venoms are a valuable source of pharmacologically active compounds; some of the peptide toxin families from the snail venoms are known to interact with potassium channels. We report the purification, synthesis, and characterization of κM-conotoxin RIIIJ from the venom of a fish-hunting species, Conus radiatus. This conopeptide, like a previously characterized peptide in the same family, κM-RIIIK, inhibits the homotetrameric human Kv1.2 channels. When tested in Xenopus oocytes, κM-RIIIJ has an order of magnitude higher affinity (IC50=33 nM) to Kv1.2 than κM-RIIIK (IC50=352 nM). Chimeras of RIIIK and RIIIJ tested on the human Kv1.2 channels revealed that Lys-9 from κM-RIIIJ is a determinant of its higher potency against hKv1.2. However, when compared in a model of ischemia/reperfusion, κM-RIIIK (100 μg/kg of body weight), administered just before reperfusion, significantly reduces the infarct size in rat hearts in vivo without influencing hemodynamics, providing a potential compound for cardioprotective therapeutics. In contrast, κM-RIIIJ does not exert any detectable cardioprotective effect. κM-RIIIJ shows more potency for Kv1.2-Kv1.5 and Kv1.2-Kv1.6 heterodimers than κM-RIIIK, whereas the affinity of κM-RIIIK to Kv1.2-Kv1.7 heterodimeric channels is higher (IC50=680 nM) than that of κM-RIIIJ (IC50=3.15 μM). Thus, the cardioprotection seems to correlate to antagonism to heteromultimeric channels, involving the Kv1.2 α-subunit rather than antagonism to Kv1.2 homotetramers. Furthermore, κM-RIIIK and κM-RIIIJ provide a valuable set of probes for understanding the underlying mechanism of cardioprotection. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.