PulseNet is a national network of pubic health and food regulatory laboratories established in the US to detect clusters of foodborne disease and respond quickly to foodborne outbreak investigations. PulseNet laboratories currently subtype Escherichia coli O157:H7, non-typhoidal Salmonella, and Shigella isolates by a highly standardized 1-day pulsed-field gel electrophoresis (PFGE), and exchange normalized DNA "fingerprint" patterns via the Internet. We describe a standardized molecular subtyping protocol for subtyping Listeria monocytogenes that was recently added to PulseNet. The subtyping can be completed within 30 h from the time a pure culture of the bacteria is obtained.