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Fractionation of venom from an agelenid spider, Tegenaria agrestis, resulted in the isolation of a family of three peptides with potent insecticidal activity. These peptide toxins, TaITX-1, -2, and -3, whose sequences were revealed from cloned cDNAs, each consist of 50 amino acid residues, six of which are cysteines. They appear to be amidated at their C-termini and exhibit greater than 90% sequence identity. Unlike other reported spider toxins, the TaI toxins are processed from precursors containing no propeptide sequences. In lepidopteran larvae and corn rootworm beetles, the insecticidal Tegenaria toxins caused an unusual excitatory symptomatology with 50% paralytic doses ranging from 0.23 to 2.6 nmol/g. In a series of electrophysiological experiments performed in house fly larvae, these toxins caused an elevated rate of firing from central nervous system neurons. No significant effects were found when any peripheral sensory or motor systems were examined. Thus, it appears that the TaI toxins may act in a fashion not previously reported for insecticidal peptide toxins; they may act directly on the insect central nervous system.