To estimate the polyamine distribution in bovine lymphocytes and rat liver, the binding constants (K) for DNA, RNA, phospholipid, and ATP were determined under the conditions of 10 mM Tris-HCl, pH 7.5, 2 mM Mg2+, and 150 mM K+. The binding constants of spermine for calf thymus DNA, Escherichia coli 16 S rRNA, phospholipid in rat liver microsomes and ATP were 1.15 x 10(2), 6.69 x 10(2), 2.22 x 10(2), and 5.95 x 10(2) M-1, respectively. From these binding constants and experimentally determined cellular concentrations of macromolecules, ATP, and polyamines, spermine distribution in the cells was estimated. In bovine lymphocytes, the mols of spermine bound to DNA, RNA, phospholipid, and ATP were 0.79, 3.7, 0.23, and 4.3 per 100 mol of phosphate of macromolecules or ATP, respectively. In rat liver, they were 0.19, 1.0, 0.05, and 0.97/100 mol of phosphate of macromolecules or ATP, respectively. The binding constants of spermidine for macromolecules and ATP were smaller than those of spermine, but a similar tendency was observed with spermidine distribution among macromolecules and ATP in the above two cells. The amount of polyamine bound to DNA and phospholipid was significantly lower than that to RNA. When either the Mg2+ or K+ concentration increased, the amount of free spermine and that bound to RNA and ATP increased, but the amount of spermine bound to DNA and phospholipid decreased. The results indicate that most polyamines exist as a polyamine-RNA complex in cells. Under the conditions that globin synthesis is stimulated by spermine in a rabbit reticulocyte cell-free system, the amount of spermine bound to RNA was very close to the value estimated in the cells.