Despite the extremely high concentration of DNA in nucleoid/nuclear regions, chromosomal dimerization and entanglement are avoided. To help understand this, we measured the effective concentration of DNA in E. coli, a value that reflects the functional impact of the cellular milieu on DNA site reactivity. We used as probes plasmid fusion reactions by two site-specific recombinases. The normalized extents and rates of fusion in these systems were much lower in vivo than in analogous in vitro reactions. We calculate that the effective concentration of plasmid DNA is about one order of magnitude lower than the chemical concentration. We suggest that in bacterial cells DNA accessibility is highly restricted and that this dominates the forces that increase DNA activity, such as macromolecular crowding.