We report the first molecular characterization of a precursor sequence for a small, Ca2+ channel blocking, peptide spider toxin, omega-agatoxin IA. By integrating information generated from a molecular genetic approach using agatoxin cDNAs with data provided from mass spectrometry of the mature toxin, we were able to deduce the likely mechanisms by which the toxin precursor peptide is processed to its mature heterodimeric form. A particularly interesting feature of the prepropeptide is the occurrence of two glutamate-rich sequences interposed between the signal sequences, the major peptide toxin, and the minor toxin peptide. Excision of the more distal glutamate-rich region appears to be signaled by flanking arginine residues but likely occurs only after a disulfide linkage has formed between the major and minor chains of the mature toxin. Our molecular genetic approach toward characterizing this toxin will allow us to quickly generate a series of spider sequences from which mature toxin structures can be deduced and eventually expressed. Additionally, this approach will provide insights into the evolutionary divergence observed among spider peptide toxins.