SignificanceBacteria replicate the genome on average once per generation, irrespective of growth condition–dependent changes in generation time and cell size. The strategy appears to be to initiate replication in a narrow chromosome-to-size ratio interval for each growth condition. To investigate howEscherichia coliaccurately compares its size to the number of chromosomes, we perturbed some of the mechanisms proposed to achieve this precision and monitored how replication initiation was affected with time-lapse single-cell fluorescence microscopy. We found that the regulatory inactivation of DnaA (RIDA) mechanism, which converts the replication initiation molecule DnaA to its inactive form in proportion to the number of chromosomes, was the most important factor and that other previously described mechanisms have a less critical role.