The emergence of genomics; ongoing computational advances; and the development of large-scale sequence, structural, and functional databases have created important new interdisciplinary linkages between molecular evolution, molecular biology, and enzymology. The five minireviews in this series survey advances and challenges in this burgeoning field from complementary perspectives. The series has three major themes. The first is the evolution of enzyme superfamilies, in which members exhibit increasing sequence, structural, and functional divergence with increasing time of divergence from a common ancestor. The second is the evolutionary role of promiscuous enzymes, which, in addition to their primary function, have adventitious secondary activities that frequently provide the starting point for the evolution of new enzymes. The third is the importance of in silico approaches to the daunting challenge of assigning and predicting the functions of the many uncharacterized proteins in the large-scale sequence and structural databases that are now available. A recent computational advance, the use of protein similarity networks that map functional data onto proteins clustered by similarity, is presented as an approach that can improve functional insight and inference. The three themes are illustrated with several examples of enzyme superfamilies, including the amidohydrolase, metallo-β-lactamase, and enolase superfamilies.