Transcription factors (TFs) regulate complex programs of gene transcription by binding to short DNA sequence motifs. Here, we introduce rtfbsdb, a unified framework that integrates a database of more than 65 000 TF binding motifs with tools to easily and efficiently scan target genome sequences. Rtfbsdb clusters motifs with similar DNA sequence specificities and integrates RNA-seq or PRO-seq data to restrict analyses to motifs recognized by TFs expressed in the cell type of interest. Our package allows common analyses to be performed rapidly in an integrated environment.