Disease-causing bacteria that become resistant most quickly to the antibiotic ampicillin do so by acquiring mutations that allow them to tolerate the antibiotic first, a new study reveals. Antibiotic resistance is a major threat to human health, and gaining insights into how bacteria develop resistance is critical for finding solutions to combat the problem. Bacteria survive antibiotic exposure either because they can become inactive when exposed to antibiotics (tolerance) or because they acquire active mechanisms to counter the antibiotic (resistance). Here, Irit Levin Reisman et al. analyzed batches of Escherichia coli strains intermittently exposed to the antibiotic ampicillin. They found that resistance emerged more quickly in strains that had already developed tolerance mutations. Using their experimental data, they calculated the probability that a resistance mutation will occur if tolerant mutations are already in place. Their analysis reveals that without tolerance, the resistance mutations would most often be lost during antibiotic treatment and that more than 100 cycles of antibiotic exposure would be needed for partial resistance mutations to become established in a population. These findings suggest that new drugs, or drug combinations, that decrease tolerance may help deter the evolution of resistance.