Traumatic brain injury (TBI) is a leading cause of death and disability in children. The current gold standards for diagnosis of TBI after closed head injury (CHI) have limitations, particularly in cases of inflicted injury. S100B is a protein that is specific to astrocytes. Serum S100B concentrations are increased in adults after CHI; there are no studies of serum S100B after CHI in children. The goal of this study was to measure the serum concentrations of S100B in children inflicted and noninflicted mild, moderate, and severe CHI. CHI severity was defined by initial Glasgow Coma Scale score. Forty-five children aged 0-13 years with mild (n = 27), moderate (n = 6), and severe (n = 12) CHI were enrolled prospectively. Blood was obtained as soon as possible after injury (range: 0.5-15.25 h) and every 12 h for up to 5 days when vascular access was available. Single control samples were obtained from 16 children aged 0-11 years with isolated long-bone fractures. Twenty-two patients (49%), including both patients with inflicted CHI, had an abnormal initial serum S100B concentration where an abnormal concentration was defined as greater than mean control concentration plus two standard deviations. S100B was detectable more than 12 h after injury only in patients with severe CHI. We conclude that serum S100B is increased in almost half of children after mild, moderate, and severe inflicted and noninflicted CHI. The increase is transient, lasting less than 12 h after injury, except in children with severe injury. Future research will focus on the possibility of using serum S100B as a screening test for inflicted CHI.