In stroke, gene transcription plays a central role in processes such as neuroinflammation and neuroregeneration. To predict new transcriptional regulatory mechanisms in cerebral ischemia, we applied a computational approach combining two kinds of information: the results of a microarray analysis in a mouse model of stroke and in silico detection of transcription factor (TF) binding sites in promoter regions of the genes on the array. By using a discriminative logistic regression model, we identified binding sites significantly associated with the up-regulation of genes. Out of 356 TF binding sites defined in TRANSFAC, we could link 32 to gene up-regulation in cerebral ischemia. These sites bind both TFs with an established and a so far unknown role in cerebral ischemia. To evaluate the results further we investigated whether two TFs, CCAAT/enhancer binding protein beta (C/EBP beta) and vitamin D receptor (VDR), are activated as predicted. Immunohistochemistry demonstrated that C/EBP beta and VDR translocated to the nucleus in cerebral ischemia. Chromatin immunoprecipitation revealed increased binding of C/EBP beta to the promoter of its target gene saa3. In addition, we found evidence for the up-regulation of VDR in brain samples from human stroke patients. These results confirm the activation of C/EBP beta and VDR in cerebral ischemia. Thus, our in silico analysis may provide additional information on transcriptional regulation in stroke and suggests several novel transcriptional programs for further exploration.