Toxins isolated from scorpion, snake, and spider venoms are valuable tools to probe the physiologic function and structure of ion channels. In this study, we have isolated three new toxins (heteropodatoxins) from the venom of a spider, Heteropoda venatoria. These toxins are structurally similar peptides of 29 to 32 amino acids and share sequence homology with hanatoxins isolated from the venom of a Chilean tarantula. The heteropodatoxins prolonged the action-potential duration of isolated rat ventricular myocytes, suggesting that the peptides block K+ currents. The effect of toxins on cardiac K+ currents were studied using voltage clamp techniques. The toxins blocked the transient outward K+ current but not other K+ currents in isolated rat cardiac myocytes. The mechanism of block was studied further using Kv4.2, a cloned channel believed to underlie transient outward K+ current in rat myocytes. The toxins blocked Kv4.2 current expressed in Xenopus laevis oocytes in a voltage-dependent manner, with less block at more positive potentials. In addition, the toxins slowed the time course of current activation and inactivation and shifted the voltage dependence of current inactivation to more positive potentials. The heteropodatoxins represent new pharmacologic probes to study the role of Kv4.2 channels in cardiac and neural tissue.