A TCEP (tris(2-carboxyethyl)phosphine)-based reduction/cysteine amidoalkylation strategy was utilized to solve the disulfide structures of omega-agatoxins IVB (1) and TVC (2). These p-type calcium channel antagonists, isolated from the American funnel-web spider Agelenopsis aperta, were found to have the same amino acid sequence and disulfide bond motif. The difference between omega-Aga IVB (1) and IVC (2) resides in the C-termini (Ser(46)) of both peptides. omega-Aga IVB (1) contains a D-serine residue while omega-Aga TVC (2) has an L-serine in this position. The existence of D-amino acids in eucaryotic systems is extremely rare. To our knowledge, however, this is the first time that a peptide sequence with an established cystine pattern possesses an amino acid in both D and L configurations.