The carboxyl-terminal sequences of the two polypeptide chains of the Saccharomyces cerevisiae K1 killer toxin were determined by protein sequencing and amino acid analysis of peptide fragments generated from the mature, secreted toxin. The COOH-terminal amino acid of the beta chain is histidine 316, the final residue encoded by the precursor gene. The COOH terminus of the alpha chain is at alanine 147 of the preprotoxin. Amino acid composition data for the purified toxin are consistent with that predicted from the gene sequence of the preprotoxin where the alpha and beta subunits consist of amino acid residues 45-147 and 234-316, respectively. The molecular weight of the mature alpha beta dimer is about 20,658. The COOH-terminal sequence determination completes the location of the toxin subunits in the precursor, and its configuration may be represented as prepropeptide-Pro-Arg-alpha-Arg-Arg-gamma-Lys-Arg-beta, where gamma represents the interstitial glycosylated peptide. The COOH terminal side of the paired basic residues (Arg-148 Arg-149 and Lys-232 Arg-233 of preprotoxin) are endoproteolytic processing sites for the product of the KEX2 gene (Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984) Cell 37, 1075-1089), and thus maturation of the alpha subunit of killer toxin apparently requires a carboxypeptidase B-like activity. A possible candidate for this activity is the product of the KEX1 gene (Dmochowska, A., Dignard, D., Henning, D., Thomas, D.Y., and Bussey, H. (1987) Cell, in press).