We describe the first potent and selective blocker of the class E Ca²⁺channel. SNX-482, a novel 41 amino acid peptide present in the venom of the African tarantula, Hysterocrates gigas, was identified through its ability to inhibit human class E Ca²⁺ channels stably expressed in a mammalian cell line. An IC₅₀ of 15−30 nM was obtained for block of the class E Ca²⁺ channel, using either patch clamp electrophysiology or K⁺-evoked Ca²⁺ flux. At low nanomolar concentrations, SNX-482 also blocked a native resistant or R-type Ca²⁺ current in rat neurohypophyseal nerve terminals, but concentrations of 200−500 nM had no effect on R-type Ca²⁺ currents in several types of rat central neurons. The peptide has the sequence GVDKAGCRYMFGGCSVNDDCCPRLGCHSLFSYCAWDLTFSD-OH and is homologous to the spider peptides grammatoxin S1A and hanatoxin, both peptides with very different ion channel blocking selectivities. No effect of SNX-482 was observed on the following ion channel activities:  Na⁺ or K⁺ currents in several cultured cell types (up to 500 nM); K⁺ current through cloned potassium channels Kv1.1 and Kv1.4 expressed in Xenopus oocytes (up to 140 nM); Ca²⁺ flux through L- and T-type Ca²⁺ channels in an anterior pituitary cell line (GH3, up to 500 nM); and Ba²⁺ current through class A Ca²⁺ channels expressed in Xenopus oocytes (up to 280 nM). A weak effect was noted on Ca²⁺ current through cloned and stably expressed class B Ca²⁺ channels (IC₅₀ > 500 nM). The unique selectivity of SNX-482 suggests its usefulness in studying the diversity, function, and pharmacology of class E and/or R-type Ca²⁺ channels.