The α/β barrel fold is adopted by most enzymes performing a variety of catalytic reactions, but with very low sequence similarity. In order to understand the stabilizing interactions important in maintaining the α/β barrel fold, we have identified residue clusters in a dataset of 36 α/β barrel proteins that have less than 10% sequence identity within themselves. A graph theoretical algorithm is used to identify backbone clusters. This approach uses the global information of the nonbonded interaction in the α/β barrel fold for the clustering procedure. The nonbonded interactions are represented mathematically in the form of an adjacency matrix. On diagonalizing the adjacency matrix, clusters and cluster centers are obtained from the highest eigenvalue and its corresponding vector components. Residue clusters are identified in the strand regions forming the β barrel and are topologically conserved in all 36 proteins studied. The residues forming the cluster in each of the α/β protein are also conserved among the sequences belonging to the same family. The cluster centers are found to occur in the middle of the strands or in the C-terminal of the strands. In most cases, the residues forming the clusters are part of the active site or are located close to the active site. The folding nucleus of the α/β fold is predicted based on hydrophobicity index evaluation of residues and identification of cluster centers. The predicted nucleation sites are found to occur mostly in the middle of the strands. © 2001 Wiley-Liss, Inc.