Phosphoglycerate kinase (PGK) catalyzes the reversible phosphoryl transfer between 1,3-bisphosphoglycerate and ADP to form 3-phosphoglycerate and ATP in the presence of magnesium. The detailed positions of the substrates during catalysis have been a long-standing puzzle due to the major conformational changes required for active site formation. Here we report the refined closed form Trypanosoma brucei PGK ternary complex at an improved resolution of 2.5 A, together with the crystal structure of closed form T. brucei PGK in complex with the nucleotide analogue AMP-PNP. In the 180 000 Da asymmetric unit of the ternary complex, four closed form PGK molecules appear to be arranged as two asymmetric dimers. Quite surprisingly, each dimer is comprised of one 3-phosphoglycerate. MgADP.PGK ternary complex and one Pi.MgADP.PGK pseudoternary complex. The substrates in the ternary complex are bound in a fashion nearly identical to that in open form PGK, but a 30 degrees hinge bending conformational change has brought them together and in-line for catalysis. The pseudoternary complex subunits exhibit a similar hinge closure but contain, instead of 3-phosphoglycerate, a single phosphate molecule bound in the active site. This phosphate binds to a site expected for the 1-position phosphate of 1,3-bisphosphoglycerate, hence providing information for the binding mode for this chemically unstable substrate. The structure of the binary PGK.MgAMP-PNP complex indicates the binding mode for MgATP. An examination of the interactions made by the transferring phosphate groups of the substrate, 1, 3-bisphosphoglycerate, and the product, ATP, reveals that in each case only two of the three nonbridging phosphate oxygens are stabilized by hydrogen bonds. In contrast, a model of the transition state phosphoryl group based on all available structural data reveals active site stabilization of all three negatively charged phosphoryl oxygens. These structural models provide insight into the nature of the phosphoryl-transfer reaction catalyzed by PGK and related enzymes.