The diagnosis of postsurgical mediastinitis (PSM) among patients with sternal wound complication (SWC) after cardiac surgery is sometimes difficult, as fever, elevated C-reactive protein levels, and chest pain can be caused by a general inflammatory reaction to the operative trauma and/or sternal dehiscence without infection. The definitions of PSM usually used emphasize clinical signs and symptoms easily observed by the surgeon. The aim of the study was to investigate whether the use of standardized multiple tissue sampling, optimal culturing methods, and strain typing, together with a microbiological criterion for infection, could identify more infected patients than clinical assessment alone. Patients reexplored due to SWC after cardiac artery bypass grafting (CABG) or heart valve replacement (HVR) with or without CABG performed at the Department for Cardio-Thoracic Surgery at the Uppsala University Hospital between 10 March 1998 and 9 September 2000 were investigated prospectively. Tissue samples were taken from the sternum or adjacent mediastinal tissue, preferably before the administration of antibiotics. Culturing was performed both directly (on agar plates) and using enrichment broth. Species identification was performed by standard methods, and strain typing was performed by pulsed-field gel electrophoresis. A total of 41 cases with at least five tissue samples each were included in the study group. Of these patients, 32 were infected according to the microbiological criterion (i.e., the same strain was found in >/=50% of the samples). Staphylococcus epidermidis was the primary pathogen in 38% of the cases (12/32), S. aureus was the primary pathogen in 31% (10/32), P. acnes was the primary pathogen in 25% (8/32), and S. simulans and S. haemolyticus were the primary pathogens in 3% (1/32) each. All cases of S. aureus infection and 86% (12/14) of coagulase-negative staphylococcus (CoNS) infections were identified from primary cultures. All cases fulfilling the microbiological criterion for S. aureus infection were clinically diagnosed as cases of infection, but among the 14 cases fulfilling the criterion for microbiological diagnosis of CoNS infection, only 10 appeared to qualify clinically as cases of infection. Among the patients with sternal dehiscence in whom a microbiological diagnosis was established, 67% (12/18) had a CoNS infection, compared to 14% (2/14) of those without sternal dehiscence. The difference was statistically significant. PSM caused by S. aureus is readily identified by the surgeon, whereas 30% of cases with CoNS infections may be misinterpreted as noninfected. Multiple sampling before administration of antibiotics, primary culturing on agar plates, species identification, strain typing, and susceptibility testing should be used to ensure a fast and microbiologically correct diagnosis which identifies the primary pathogen and infected patients among those with minor infective symptoms. The role of P. acnes as a possible cause of PSM needs further investigation. PSM caused by CoNS is significantly related to sternal dehiscence.