The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U) ] by polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2. 7. 7. 8] from Micrococcus luteus have been studied. Although the breakdown of all the synthetic polynucleotides tested was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C)>poly(A)>poly(U) at pH 7.5. However, the difference in degree of stimulation among polynucleotides decreased as the pH or monovalent cation concentration was increased. In the presence of heparin, an inhibitor of polynucleotide phosphorylase hydrolysis of polynucleotides, spermidine clearly stimulated the breakdown of poly(C) and poly(A), while the breakdown of poly(U) was stimulated only slightly by the addition of spermidine. Although binding of [¹⁴C]spermine to polynucleotide phosphorylase was observed by gel filtration, the amount of spermine bound to the enzyme was much less than that to RNA.