Traditionally, the classification of beta-lactamases has followed the general principles of describing enzymes by the characterization of their activity against substrates. New types of beta-lactam structures with therapeutic relevance have emerged, necessitating more specific substrate profiles. Biophysical characterization, i.e., primarily isoelectric focusing, has greatly improved classification, particularly of nonchromosomal beta-lactamases. The classification of beta-lactamases of both gram-positive and gram-negative organisms is reviewed and updated by adding data on recently discovered enzymes. Data on amino acid sequence and molecular structure that are available for an increasing number of beta-lactamases will gradually be incorporated into the established system. In addition, the introduction of evolutionary aspects should serve to modify the existing system, which to date has been based on arbitrarily selected characteristics.