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<p>Abstract</p> <p>Background</p> <p>Combinatorial regulation of transcription factors (TFs) is important in determining the complex gene expression patterns particularly in higher organisms. Deciphering regulatory rules between cooperative TFs is a critical step towards understanding the mechanisms of combinatorial regulation.</p> <p>Results</p> <p>We present here a Bayesian network approach called GBNet to search for DNA motifs that may be cooperative in transcriptional regulation and the sequence constraints that these motifs may satisfy. We showed that GBNet outperformed the other available methods in the simulated and the yeast data. We also demonstrated the usefulness of GBNet on learning regulatory rules between YY1, a human TF, and its co-factors. Most of the rules learned by GBNet on YY1 and co-factors were supported by literature. In addition, a spacing constraint between YY1 and E2F was also supported by independent TF binding experiments.</p> <p>Conclusion</p> <p>We thus conclude that GBNet is a useful tool for deciphering the "grammar" of transcriptional regulation.</p>