This study investigates the DNA binding specificities of 104 distinct mouse transcription factors (TFs) using protein binding microarrays (PBMs). The authors found that each TF analyzed had unique binding preferences, with approximately half recognizing multiple distinct sequence motifs. This complexity in DNA recognition is significant for gene regulation and the evolution of transcriptional regulatory networks. The study also identified secondary motifs for nearly half of the TFs, which can be bound nearly as well as primary motifs. Additionally, the authors observed position interdependence in binding sites, where specific nucleotide positions are strongly correlated. The findings highlight the richness and diversity in DNA binding preferences, which have implications for understanding protein-DNA interactions and genome analysis. The data also suggest that multiple motif models may be more accurate for representing TF binding profiles than single motif models.This study investigates the DNA binding specificities of 104 distinct mouse transcription factors (TFs) using protein binding microarrays (PBMs). The authors found that each TF analyzed had unique binding preferences, with approximately half recognizing multiple distinct sequence motifs. This complexity in DNA recognition is significant for gene regulation and the evolution of transcriptional regulatory networks. The study also identified secondary motifs for nearly half of the TFs, which can be bound nearly as well as primary motifs. Additionally, the authors observed position interdependence in binding sites, where specific nucleotide positions are strongly correlated. The findings highlight the richness and diversity in DNA binding preferences, which have implications for understanding protein-DNA interactions and genome analysis. The data also suggest that multiple motif models may be more accurate for representing TF binding profiles than single motif models.