Sequence-specific protein-DNA interactions (PDIs) are critical for regulating many cellular processes, including transcription, DNA replication, repair, and rearrangement. We review recent experimental advances in high-throughput technologies designed to characterize PDIs and discuss recent studies that use these tools, including ChIP-chip/seq, SELEX-based approaches, yeast one-hybrid, bacterial one-hybrid, protein binding microarray, and protein microarray. The results of these studies have challenged some long-standing concepts of PDI and provide valuable insights into the complex transcriptional regulatory networks.