Bacillus cereus sphingomyelinase (Bc-SMase) belongs to the Mg2+-dependent neutral sphingomyelinase (nSMase) which hydrolyzes sphingomyelin (SM) to produce phosphocholine and ceramide, and acts as an extracellular hemolysin. Bc-SMase has two metal ion-binding sites in a long horizontal cleft across the molecule, with one Mg2+ in the central region of the cleft and one divalent metal ion at the side-edge of the cleft. The role of the Mg2+ at the side-edge of the long horizontal cleft in Bc-SMase remains unresolved. The replacement of Asn-57, Glu-99, and Asp-100 located in close proximity to Mg2+ at the side-edge with alanine resulted in a striking reduction in binding to and hydrolysis of sphingomyelin in membranes of sheep erythrocytes or SM-liposomes but that of Phe-55 did not. However, the replacement of these residues had little effect on the enzymatic activity. N57A, E99A, and D100A contained 2 mol of Mg2+. per mol of protein, and the wild type and F55A contained 3 mol. A crystal analysis showed that N57A with Mg2+ had no metal ion at the side-edge. These results indicate that the Mg2+ at the side-edge of Bc-SMase plays an important role in the binding to membranes. Published by Elsevier Inc.