The purpose of this study was to evaluate the value of real-time molecular typing of Shiga toxin (Verocytotoxin)-producing Escherichia coli (STEC) infections in order to detect possible outbreaks of infections. All laboratory confirmed STEC infections in Denmark from 2003 to mid 2005 were routinely characterized by serotyping, virulence genes characterization, and subtyping by pulsed-field gel electrophoresis (PFGE) using the PulseNet protocol for STEC O157. The study included 312 STEC isolates representing 50 different O groups and 75 O:H-serotypes, and 68% of the isolates belonged to the eight most common O-groups: O157 (26%), O103 (13%), O146 (8%), O26 (8%), O117 (4%), O145 (3%), O128 (3%), and O111 (2%). The remaining O-groups constituted less than 2% each, and 8.1% of the isolates were O-rough. The eae gene was found in 60% of all isolates, and detection of the two main Shiga toxin genes showed that 40% had stx1 only, 31% had stx2 only, and 29% had both stx1 and stx2. A high diversity was seen within all O groups, and for most of the rare O groups, the number of PFGE profiles equaled the number of isolates. However, one outbreak of E. coli O157 was detected by the routine PFGE typing. The value of "real-time' PFGE typing of the infrequent serotypes is limited if the full scheme for O-grouping or O:H-serotyping is used routinely for all STEC isolates. Possible outbreaks can then be detected by the increased number of isolates within a particular serotype. PFGE typing would then be valuable in subsequent steps of the outbreak investigation. However, routine PFGE typing of the three to five most common O groups will enable early recognition of possible outbreaks.