Eight linear cationic peptides with cytolytic and insecticidal activity, designated cyto-insectotoxins (CITs), were identified in Lachesana tarabaevi spider venom. The peptides showed antibiotic activity towards Gram-positive and Gram-negative bacteria at micromolar concentrations as well as toxicity to insects. The primary structures of the toxins were established by direct Edman sequencing in combination with enzymatic and chemical polypeptide degradation and MS. CITs represent a novel class of cytolytic molecules and spider venom toxins. They are the first example of molecules showing equally potent antimicrobial and insecticidal effects. Analysis of L. tarabaevi venom gland expressed sequence tag database revealed the primary structures of the protein precursors; eight peptides homologous with the purified toxins were additionally predicted. CIT precursors share a conventional prepropeptide structure with an acidic prosequence and a processing motif common to most spider toxin precursors. The most abundant peptide, CIT 1a, was chemically synthesized, and its lytic activity on different bacterial strains, human erythrocytes and lymphocytes, insect cells, planar lipid bilayers and lipid vesicles was characterized. The spider L. tarabaevi is suggested to have evolved to rely on a unique set of linear cytolytic toxins, as opposed to the more common disulfide-containing spider neurotoxins.