In patients suffering from cerebrovascular diseases and traumatic brain damage, increases in serum levels of protein S100B are positively correlated with the severity of the insult. Since high concentrations of S100B have been shown to exert neurotoxic effects, the objective of this study was to characterize the regulatory mechanisms underlying control of S100B release from astrocytes. To that end, we analyzed the kinetics and amount of S100B release in correlation with regulation of S100B gene expression in an in vitro ischemia model. Astrocyte cultures were treated with combined oxygen, serum and glucose deprivation, serum and glucose deprivation or hypoxia alone for 6, 12 and 24 h, respectively. While oxygen, serum and glucose deprivation triggered the most rapid release of S100B, serum and glucose deprivation provoked comparable levels of released S100B at the later time points. In contrast to oxygen, serum and glucose deprivation and serum and glucose deprivation, hypoxia alone elicited only marginal increases in secreted S100B. Parallel analysis of extracellular lactate dehydrogenase and the number of viable cells revealed only moderate cell death in the cultures, indicating that S100B was actively secreted during in vitro ischemia. Interestingly, S100B mRNA expression was potently downregulated after 12 and 24 h of oxygen, serum and glucose deprivation, and prolonged oxygen, serum and glucose deprivation for 48 h was associated with a significant reduction of S100B release at later time intervals, whereas lactate dehydrogenase levels remained constant. Our data suggest that secretion of S100B during the glial response to metabolic injury is an early and active process.