A disc diffusion method, based on the idea of Klundert, for screening of substrate profiles of beta-lactamases was developed in order to perform epidemiological studies. The method was tested against 30 different reference beta-lactamases and 59 clinical isolates known to produce TEM-1, SHV-1 and BRO-1. The reproducibility and discriminating power of the disc diffusion method made it possible to differentiate between eight types of substrate profiles for the 30 reference beta-lactamases and to differentiate between TEM-1, SHV-1 and BRO-1 from clinical isolates. In combination with analytical isoelectric focusing the disc diffusion method gives a reliable identification of beta-lactamases.