Serine racemase (SR) is a brain enzyme present in glial cells where it isomerises L-serine into D-serine, that in turn, diffuses and coactivates the NMDA receptor through the binding to the so-called "glycine site". We have developed a method for the slow expression of SR in an eukaryotic vector that permits the correct insertion of the prosthetic group into the active site, rendering functional SR with a Km towards L-serine of 4.8 mM. Divalent cations such as calcium or manganese were necessary for complete enzyme activity, whereas the presence of chelators such as EDTA completely inhibited the enzyme. Moreover, direct binding of calcium to SR was evidenced using 45Ca2+. Gel filtration of the recombinant SR revealed the protein to be in a dimer-tetramer equilibrium. Addition of EDTA to a calcium-saturated serine racemase evokes a profound conformational change, as monitored by both fluoresence and circular dichroism techniques. Fluorescence titration allowed us to calculate a binding constant for calcium of 6.2 mM. Reagents that react with sulfhydril groups, such as cystamine, were potent inhibitors of SR, in clear reflection that one or more cysteine residues are important for enzyme activity. Additionally, sixteen Serine analogues were tested as a putative SR substrate or inhibitors. Significant inhibition was only observed for L-Ser-O-sulfate, L-cycloserine and L-cysteine. Finally, activation of brain SR as a result of the changes in calcium concentration was studied in primary astrocytes. Treatment of astrocytes with the calcium ionophore A23187, as well as with compounds that augment the intracellular calcium levels such as glutamate or kainate lead to an increase in the amount of D-serine present in the extracellular medium. These results suggest that there might be a glutamatergic-mediated regulation of SR activity by intracellular calcium concentration.