Persisters are suggested to be the products of a phenotypic variability that are quasi-dormant forms of regular bacterial cells highly tolerant to antibiotics. Our previous investigations revealed that a decrease in antibiotic tolerance of Escherichia coli cells could be reached through the inhibition of key enzymes of polyamine synthesis (putrescine, spermidine). We therefore assumed that polyamines could be involved in persister cell formation. Data obtained in our experiments with the polyamine-deficient E. coli strain demonstrate that the formation of persisters tolerant to netilmicin is highly upregulated by putrescine in a concentration-dependent manner when cells enter the stationary phase. This period is also accompanied by dissociation of initially homogenous subpopulation of persister cells to some fractions differing in their levels of tolerance to netilmicin. With three independent experimental approaches, we demonstrate that putrescine-dependent upregulation of persister cell formation is mediated by stimulation of rpoS expression. Complementary activity of putrescine and RpoS results in ~ 1000-fold positive effect on persister cell formation.