Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and PGHS-2) catalyze the first step in the biosynthetic pathway that produces prostaglandins and thromboxanes. The fatty acid endoperoxide/hydroperoxide substrate, PGG(2), binds on the distal side of the heme that forms the peroxidase (POX) site of PGHSs and generates the alcohol PGH(2), by cleaving the oxygen-oxygen bond of the 15-hydroperoxide group. The structure of the POX site of PGHS, as with other peroxidases, includes the invariant distal histidine residue His207 and a glutamine, Gln203. We report the first molecular dynamics (MD) simulation of a PGG(2) analogue (pseudo-PGG(2)) bound to the peroxidase site of PGHS-1; pseudo-PGG2 lacks the endoperoxide group and double bonds of PGG(2) but is otherwise identical to PGG(2). In the MD of the substrate-free state of PGHS-1, a water migrated to the heme active site to become the sixth ligand of the iron, and in time, it hydrogen bonded to other waters, forming chains that extended into the bulk solvent. A location for pseudo-PGG(2) was found by scanning for low van der Waals contact energies in the distal heme pocket, and its geometry was refined by Simulated annealing, but the resulting position was still not close enough to the iron or His207 to support catalysis. During a 2-ns MD simulation of the complex, the hydroperoxide oxygens move to within hydrogen-bonding distance of His207 and Gln203 and ligate to the heme iron, positioning the substrate appropriately for peroxidase catalysis. Water is excluded from the active site by this large substrate.