The secondary and the tertiary structures of beta-galactosidase from Lactococcus lactis ssp. lactis 7962 were designed by Nnpredict and Sybyl Version 6.3. Structural modeling of beta-galactosidase has shown that Glu-384 and Glu-429 are ligands for Mg2+ and Mg2+ is required for maximum activity. To confirm this prediction, we generated seven site specific mutants: Glu-384-Gln; Glu-384-Val; His-386-Phe; Asn-428-Asp; Glu-429-Gln; 384Gln-429Gln and 384Val-429Gln. The beta-galactosidases substituted at Glu-384 or Glu-429 had < 1% of the activity of the native enzyme with ONPG as substrate. The substitution of Glu-384 or Glu-429, which removed only one of the coordinating ligand for Mg2+, was still affected by Mg2+, but the mutants 384Gln-429Gln or 384Val-429Gln, which had been modified both Mg2+-binding sites, were not affected by Mg2+. Thus, Glu-384 and Glu-429 are probably ligands of Mg2+ and the three dimensional disposition of Mg2+ and its neighborhood interactions (Glu-384, Glu-429, Asp-428 or His-386) are important in the maintenance of beta-galactosidase activity.