The transcriptional repressor CcpN from Bacillus subtilis mediates the CcpA-independent catabolite repression of three genes, sr1, encoding a small regulatory RNA, and two gluconeogenesis genes, gapB and pckA. The intracellular concentration of CcpN was determined to be around 4000 molecules per cell. The B. subtilis genome was scanned for potential new CcpN target genes, out of which three showed CcpN-binding activity in their upstream region. EMSAs (electrophoretic mobility shift assays) demonstrated that the promoter regions of two putative targets, thyB encoding thymidylate synthase B and yhaM encoding a 5'-3' exo-RNAse, bound CcpN with significant affinity. A detailed contact probing of CcpN-DNA interactions revealed an interesting new binding pattern at the thyB promoter, where the whole promoter appears to be contacted by CcpN. Using lacZ-reporter gene fusions and in vitro transcription assays, the thyB promoter was investigated for a regulatory effect of CcpN. Surprisingly, CcpN does not repress transcription at this promoter, but instead acts as an activator. Alignments of the thyB promoters of different Gram-positive bacteria encoding CcpN revealed CcpN consensus-binding sites in a significant number of them. Our data show that a bioinformatics-based approach combined with in vivo and in vitro experiments can be used to identify new targets of transcriptional regulators.