A set of 49 high-resolution (<or=2.2 A) structures of the TEM, SHV, and CTX-M class A beta-lactamase families was systematically analyzed to investigate the role of conserved water molecules in the stabilization of the Omega-loop. Overall, 13 water molecules were found to be conserved in at least 45 structures, including two water positions which were found to be conserved in all structures. Of the 13 conserved water molecules, 6 are located at the Omega-loop, forming a dense cluster with hydrogen bonds to residues at the Omega-loop as well as to the rest of the protein. This layer of conserved water molecules is packed between the Omega-loop and the rest of the protein and acts as structural glue, which could reduce the flexibility of the Omega-loop. A correlation between conserved water molecules and conserved protein residues could in general not be detected, with the exception of the conserved water molecules at the Omega-loop. Furthermore, the evolutionary relationship between the three families, derived from the number of conserved water molecules, is similar to the relationship derived from phylogenetic analysis.