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Mild head injury (MHI) implies a risk for traumatic brain injury and even a small risk for development of an intracranial hematoma. Head computed tomography (CT) is recommended for early detection of such pathologic findings. The present multicenter study was performed to investigate whether determination of protein S100B in serum could contribute to the selection of patients for CT scanning.We included 226 patients with a history of head injury and a Glasgow Coma Scale (GCS) score of 13 to 15 at admission to hospital. Blood samples for S100B analysis and head CT were obtained within 12 hours after the injury. The diagnostic properties of S100B measurements for prediction of intracranial injury revealed by CT were tested with receiver operating characteristic (ROC) analysis and cross-table analysis at different cut-off levels. We also included analysis of S100B levels normalized to correspond to blood sampling 1 hour after the injury.CT showed intracranial injury in 21 (9.3%) patients. S100B levels were significantly (p < 0.001) elevated in patients with intracranial injury (mean, 0.36; 95% CI, 0.21–0.50 μg/L) compared with those in patients without intracranial injury (mean, 0.18; 95% CI, 0.16–0.20 μg/L). ROC curve analysis showed a significant (p = 0.001) area under the curve (0.73; 95% CI, 0.62–0.84). Cross-table analysis showed that 20 of 21 (sensitivity 0.95) patients with intracranial injury were detected at a cut-off level of 0.10 μg/L, but 141 of 205 (specificity 0.31) patients with no such injury also had a S100B level above this limit. Exclusion of cases with blood samples collected more than 3 hours after injury or normalization did not improve the diagnostic properties.Determination of serum S100B cannot replace the clinical examination or use of CT for patients with minor head injury, but adding S100B measurement to the clinical evaluation might support selection of patients for CT scanning.