Pig muscle phosphoglycerate kinase has been crystallized from polyethyleneglycol in the presence of its substrate 3-phospho-D-glycerate (3-PG) and the structure has been determined at 2.0 A resolution. The structure was solved using the known structure of the substrate-free horse muscle enzyme and has been refined to a crystallographic R-factor of 21.5%. 3-Phospho-D-glycerate is bound to the N-domain of the enzyme through a network of hydrogen bonds to a cluster of basic amino acid residues and by electrostatic interactions between the negatively charged phosphate and these basic protein side chains. This binding site is in good agreement with earlier proposals [Banks et al., Nature (London) 279:773-777, 1979]. The phosphate oxygen atoms are hydrogen bonded to His-62, Arg-65, Arg-122, and Arg-170. The 2-hydroxyl group, which defines the D-isomer of 3PG, is hydrogen bonded to Asp-23 and Asn-25. The carboxyl group of 3-PG points away from the N-domain towards the C-domain and is hydrogen bonded via a water molecule to main chain nitrogen atoms of helix-14. The present structure of the 3-PG-bound pig muscle enzyme is compared with the structure of the substrate-free horse enzyme. Major changes include an ordering of helix-13 and a domain movement, which brings the N-domain closer to the ATP-binding C-domain. This domain movement consists of a 7.7 degree rotation, which is less than previously estimated for the ternary complex. Local changes close to the 3-PG binding site include an ordering of Arg-65 and a shift of helix-5.