Significance Gene expression is controlled by sequence-specific transcription factors (TFs), which bind to regulatory sequences in DNA. The degree to which the arrangement of motif sites within regulatory elements determines their function remains unclear. Here, we show that the positional distribution of TF motif sites within nucleosome-depleted regions of DNA fall into six distinct classes. These patterns are highly consistent across cell types and bring together factors that have similar functional and binding properties. Furthermore, the position of motif sites appears to be related to their known functions. Our results suggest that TFs play distinct roles in forming a functional enhancer, facilitated by their position within a regulatory sequence.