Chymotrypsin and other members of the serine protease enzyme family have a structure built from two similar domains, each of which is a hydrogen-bonded barrel, containing six antiparallel strands of beta-sheet bonded in the order ABCFED-A …. The folding patterns of the domains have been re-examined by several newly improved shape comparison methods to see whether the barrels could have evolved by gene duplication, as proposed by Matthews and Blow (Birktoft & Blow, 1972). The domains have a similar hydrogen-bond pattern, the same shear number (defined in this paper) for the twist of the barrel, and the cores of their β-sheets can be superimposed so that 46 topologically equivalent α-carbons fit within a root-mean-square distance of 2.43 A and a larger set of 57 α-carbons fit within 3.4 A. These results are highly significant when judged against shape comparisons of many other proteins with themselves, and give strong evidence for gene duplication. The duplication does not include any SS bridges. Both domains have a surprisingly symmetrical structure of two halves ABC, DEF paired round a dyad axis, and the half-domains are each made of two loops twisted in an L-shape, since the second strand (B or E) is bent into two halves B1, B2 or E1, E2. The cores of the four half-domains, each of 23 α-carbons, superimpose in pairs with root-mean-square distances ranging from 1.79 to 2.45 A. In the entire molecule the half-domains are related by a screw dyad which converts domain I strands (ABC) (DEF) into domain II strands (DEF) (ABC) superimposing the six strands with a root-mean-square distance of 2.35 A. These observations suggest that the Chymotrypsin barrel originally evolved from a closely-linked dimer of two intertwined half-domains which became united into one. domain by gene duplication. The enzyme evolved from a second dimer of two full domains and a second duplication. The bacterial protease B from Streptomyces griseus shows the same structural repeats and is consistent with the gene duplication hypothesis. Improved methods for shape comparison of proteins have been developed which are very fast and reliable.