Primary objectives : To examine the diagnostic value of S100 in mild traumatic brain injury (MTBI). Research design : Prospective cohort study. Methods and procedures : S100B, S100A1B and S100BB concentrations were examined in sera from patients with MTBI with an arrival Glasgow Coma Scale score of 15 or 14, patients with orthopaedic injuries and non-injured subjects. Main outcome and results : Mean values and proportions of subjects above cut-off limits for S100B and S100A1B were significantly higher in each trauma group than in non-injured controls, but only for S100A1B when patients with MTBI were compared with controls with orthopaedic injuries. Using a 97.5 percentile cut-off limit, the sensitivity of S100A1B for MTBI vs orthopaedic injury was 61% (95% confidence interval (CI) 49-73%), specificity 77% (95% CI 62-93%). The area under the ROC curve did not approach 0.9 for any cut off limit. Conclusions : Diagnostic validity of S100 in acute MTBI was not demonstrated. S100A1B has merits for long-term prognostic studies.