Eukaryotic transcriptional regulation requires the integration of complex signals by the transcriptional promoter. Distinct sequence elements, characteristic chromatin modifications and coordinated protein-DNA interactions at these sequences constitute a transcriptional regulatory code that remains poorly understood today. Here, we review recent experimental and computational advances that have enabled the identification and analysis of transcriptional promoters on an unprecedented scale, laying a foundation for systematic determination of the transcriptional regulatory networks in eukaryotic cells. The knowledge gained from these large-scale investigations has challenged some conventional concepts of promoter structure and function, and provided valuable insights into the complex gene regulatory mechanisms in a variety of organisms.