Bacterial populations produce persister cells that neither grow nor die in the presence of microbicidal antibiotics. Persisters are largely responsible for high levels of biofilm tolerance to antimicrobials, but virtually nothing was known about their biology. Tolerance of Escherichia coli to ampicillin and ofloxacin was tested at different growth stages to gain insight into the nature of persisters. The number of persisters did not change in lag or early exponential phase, and increased dramatically in mid-exponential phase. Similar dynamics were observed with Pseudomonas aeruginosa (ofloxacin) and Staphylococcus aureus (ciprofloxacin and penicillin). This shows that production of persisters depends on growth stage. Maintaining a culture of E. coli at early exponential phase by reinoculation eliminated persisters. This suggests that persisters are not at a particular stage in the cell cycle, neither are they defective cells nor cells created in response to antibiotics. Our data indicate that persisters are specialized survivor cells.