Research in the field of gene regulation is evolving rapidly in the ever-changing scientific environment. Advances in microarray techniques and comparative genomics have enabled more comprehensive studies of regulatory genomics. The study of genomic binding locations of transcription factors has enabled a more comprehensive modeling of regulatory networks. In addition, complete genomic sequences and comparison of numerous related species have demonstrated the conservation of non-coding DNA sequences, which often provide evidence for cis-regulatory binding sites. Systematic methods to decipher the regulatory mechanism are also crucial for corroborating these regulatory networks; key to these methods are motif discovery algorithms that can help predict cis-regulatory elements. These DNA-motif discovery programs are becoming more sophisticated and are beginning to leverage evidence from comparative genomics. These topics and more were discussed at the 3rd Annual RECOMB Workshop on Regulatory Genomics, which brought together more than 90 attendees and included about 22 excellent talks from leading researchers in the field. This proceedings volume contains ten selected, original manuscripts that were presented during the workshop.