Summary Amino acid analysis of the B- and C-chains of α-chymotrypsin (EC 3.4.4.5) indicated some rather serious errors in previous studies on the primary structure of bovine chymotrypsinogen A. The amino acid sequence of the C-chain has therefore been investigated further. 1. A major difficulty encountered during this investigation was the separation of the tryptophan peptides since chromatography on Dowex-1 X2 or electrophoresis-chromatography on paper was not adequate. Aromatic peptides and amino acids are eluted more slowly from dextran gels (Sephadex G25 in 0.2 M acetic acid) than non-aromatic peptides of similar length. After this preliminary separation, the aromatic peptides were separated by paper electrophoresis and chromatography. 2. Tryptic hydrolysis (trypsin, EC 3.4.4.4) of the S-sulfo C-chain resulted in a series of soluble peptides arising from the N-terminal portion of the chain and, in addition, an insoluble “core” corresponding to the C-terminal portion. The soluble peptides have been found identical with a series of peptides previously characterized in chymotrypsinogen hydrolysates by other authors and correctly positioned in the sequence. 3. The primary structure of the core was determined by: (a) characterization of 25 peptides resulting from hydrolysis by subtilopeptidase (EC 3.4.4.16), chymo-trypsin, pepsin (EC 3.4.4.1) and papain (EC 3.4.4.10); (b) characterization of DNP-peptides arising from the core N-terminus and from the sequence containing the single tyrosine residue; (c) utilization of carboxypeptidase (EC 3.4.2.1) for the C-terminal residue of the C-chain. The sequence of 42 residues finally established agrees well with the amino acid composition of the core and with the most recent structure proposed by Hartley for the C-terminal region of chymotrypsinogen A. 4. The core consists of two long peptides containing 27 and 15 residues, respectively. The insolubility of these peptides, despite the presence of several hydroxylated residues, is probably associated with a complete lack of anionic centers. This suggests that, at alkaline pH values where activation occurs and where chymotrypsins are active, the C-terminal region is probably located inside the molecules. 5. Both core peptides are linked in chain C by an Ala-Arg unit belonging to a Tyr-Ala-Arg-Val sequence. The liberation of this unit during trypsin hydrolysis suggests that even highly purified preparations of trypsin are able to split the “aromatic” bond Tyr-Ala in the C-chain.