Brain serine racemase contains pyridoxal phosphate as a prosthetic group and is known to become activated by divalent cations such as Ca(2+) and Mg(2+), as well as by ATP and ADP. In vivo, brain serine racemase is also activated by a multi-PSD-95/discs large/ZO-1 (PDZ) domain glutamate receptor interacting protein (GRIP) that is usually coupled to the GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Ca(2+) channel. In the present study, we analysed the mechanisms by which serine racemase becomes activated by GRIP, divalent cations and ATP. We show that binding of PDZ6 of GRIP to serine racemase does not result in increased d-serine production. However, full-length GRIP does augment significantly enzymatic activity. We expressed various GRIP shorter constructs to map down the regions within GRIP that are necessary for serine racemase activation. We observed that, whereas recombinant proteins containing PDZ4-PDZ5-PDZ6 are unable to activate serine racemase, other constructs containing PDZ4-PDZ5-PDZ6-GAP2-PDZ7 significantly augment its activity. Hence, activation of serine racemase by GRIP is not a direct consequence of the translocation towards the calcium channel but rather a likely conformational change induced by GRIP on serine racemase. On the other hand, the observed activation of serine racemase by divalent cations has been assumed to be a side-effect associated with ATP binding, which is known to form a complex with Mg(2+) ions. Because no mammalian serine racemase has yet been crystallized, we used molecular modelling based on yeast and bacterial homologs to demonstrate that the binding sites for Ca(2+), ATP and the PDZ6 domain of GRIP are spatially separated and modulate the enzyme through distinct mechanisms.