The crystallographic structure of a circularly permuted form of yeast PGK, 72p yPGK, has been determined to a resolution of 2.3 A by molecular replacement. In this engineered protein, the C- and N-terminal residues of the wild-type protein are directly connected by a peptide bond and new N- and C-terminal residues are located within the N-terminal domain. The overall fold of the protein is very similar to that of the wild-type protein, directly demonstrating that the continuity of a folding unit is not relevant to the folding process of the whole protein. Only limited structural changes were observed: these were in the regions associated with the new connection, in a long flexible loop in the permuted domain and in the vicinity of Arg38, a functionally important residue. The relative positions of the two domains suggested that this permuted protein adopts one of the most open/twisted conformations seen amongst PGKs of known structure. The effect of the mutation on the functional properties is more easily accounted for by a restriction of hinge-bending motion than by structural changes in the protein.