To estimate the polyamine distribution in Escherichia coli, the binding constants (K) for DNA, RNA, phospholipids, and ATP were calculated under the condition of 10 mM Tris-HCl, pH 7.5, 150 mM K+, and 10 mM Mg2+. The binding constants of spermidine for E. coli DNA, E. coli 16S rRNA, phospholipids in E. coli membrane, and ATP were 0.015, 0.066, 0.028, and 0.081 mM-1, respectively. Similarly, those of putrescine were 0.010, 0.010, 0.007, and 0.037 mM-1, respectively. The concentrations of putrescine, spermidine, and ATP and phosphates in DNA, RNA, and phospholipids in E. coli harvested at A600 = 0.3 were 32.2, 6.88, and 2.66 and 96.4, 436, and 57.2 mM, respectively. Accordingly, the percentage of spermidine bound to DNA, RNA, phospholipids, and ATP and that of free spermidine were 5.1, 90, 0.7, 0.8, and 3.8%, respectively. The percentage of putrescine bound to DNA, RNA, phospholipids, and ATP and that of free putrescine were 9.3, 48, 1.4, 2.6, and 39%, respectively. The results indicate that most spermidine exists as a spermidine--RNA complex, and about 40% and 50% of putrescine exists as a free form and a putrescine--RNA complex in cells, respectively. Under the conditions that the synthesis of specific proteins such as RNA replicase is stimulated by polyamines in a cell-free system, the amount of spermidine and putrescine bound to RNA was close to the value estimated in cells. Experiments to demonstrate the polyamine stimulation of MS2 RNA-directed RNA replicase synthesis in vivo were thus performed, and the results were confirmed.