We have developed a novel, fully automatic method for aligning the three‐dimensional structures of two proteins. The basic approach is to first align the proteins' secondary structure elements and then extend the alignment to include any equivalent residues found in loops or turns. The initial secondary structure element alignment is determined by a genetic algorithm. After refinement of the secondary structure element alignment, the protein backbones are superposed and a search is performed to identify any additional equivalent residues in a convergent process. Alignments are evaluated using intramolecular distance matrices. Alignments can be performed with or without sequential connectivity constraints. We have applied the method to proteins from several well‐studied families: globins, immunoglobulins, serine proteases, dihydrofolate reductases, and DNA methyltransferases. Agreement with manually curated alignments is excellent. A web‐based server and additional supporting information are available at http://engpub1.bu.edu/∼josephs. Proteins 2000;38:428–440. © 2000 Wiley‐Liss, Inc.