Two cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from Haemophilus parainfluenzae (HpaI), were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C-terminus of a recombinant protein. Nucleophilic attack on this thioester by the N-terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI, respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol-induced cleavage of the precursor led to the liberation of the target protein with a C-terminal thioester-tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full-length products that displayed catalytic activity indicative of the wild-type enzymes. The turnover numbers and Km for ligated and renatured RNase A were 8. 2 s-1 and 1. 5 mM, in good agreement with reported values of 8. 3 s-1 and 1. 2 mM (Hodges&Merrifield, 1975). Ligated HpaI had a specific activity of 0. 5–1. 5 × 106 U/mg, which compared favorably with the expected value of 1–2 × 106 U/mg (J. Benner, unpubl. obs. ). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.