The mode of interaction of transcription factors (TFs) on eukaryotic genomes remains a matter of debate. Single-molecule data in living cells for the TFs Sox2 and Oct4 were previously interpreted as evidence of ordered assembly on DNA. However, the quantity that was calculated does not determine binding order but, rather, energy expenditure away from thermodynamic equilibrium. Here, we undertake a rigorous biophysical analysis which leads to the concept of reciprocity. The single-molecule data imply that Sox2 and Oct4 exhibit negative reciprocity, with expression of Sox2 increasing Oct4's genomic binding but expression of Oct4 decreasing Sox2's binding. Models show that negative reciprocity can arise either from energy expenditure or from a mixture of positive and negative cooperativity at distinct genomic loci. Both possibilities imply unexpected complexity in how TFs interact on DNA, for which single-molecule methods provide novel detection capabilities.