Proteins tendency to bind to one another in a highly specific manner forming stable complexes is fundamental to all biological processes. A better understanding of complex formation has many practical applications, which include the rational design of new therapeutic agents, and the analysis of metabolic and signal transduction networks. Alanine-scanning mutagenesis made possible the detection of the functional epitopes, and demonstrated that most of the protein–protein binding energy is related only to a group of few amino acids at intermolecular protein interfaces: the hot spots. The scope of this review is to summarize all the available information regarding hot spots for a better atomic understanding of their structure and function. The ultimate objective is to improve the rational design of complexes of high affinity and specificity as well as that of small molecules, which can mimic the functional epitopes of the proteic complexes. Proteins 2007. © 2007 Wiley-Liss, Inc.