Abstract: It was previously reported that bacteriophage T4 contains the polyamines putrescine, NH2(CH2)$;IH2, and spermidine, NHQ(CH&NH(CH&NH2, in amounts sufficient to neutralize about half of the viral deoxyribonucleic acid (1). The putrescine and spermidine in the phage were found to be derived from the large amount of these polyamines normally present in the host bacterium, Escherichia coli B. It was also shown that these cations are the unidentified compounds in phage T2 reported by Hershey to be injected into the bacteria along with the viral DNA (2). In the present communication we have attempted to answer certain questions raised by these findings: 1. Is the role of the polyamines in phage that of specific or nonspecific cations for neutralizing the negatively charged phosphate groups in the DNA? 2. Are the amounts and kinds of polyamines in the phage determined by the phage or by the bacterial pool of cations? 3. Can stoichiometry between cations in the phage and the phosphate anions of the DNA be demonstrated? 4. What is the distribution of polyamines in viruses? The cations of T4 phage have been examined and a balance has been obtained between total cations and total DNA anions. The normal cation content of T4 (putrescine++, spermiclme+++, and Mg++) was changed markedly under certain conditions. When the host bacterium E. coli B was grown on minimal medium contaming spermine, NH2(CH2)3NH(CH&NH(CH&NH~, a polyamine present in animal tissues (3, 4) but not generally present in bacteria (5), the putrescine and spermidine normally present in the E. coli were replaced by spermine and acetylated spermine (6). These abnormal polyamines were found as the main polyamines in the T4 phage grown on these bacteria. The replacement of the normal polyamines suggested that the polyamines may be acting as nonspecific cations. Two types of evidence support this hypothesis. The lack of polyamines in various bacteriophages (T3, T5, P22) has been correlated with the permeability of these phages to cations; it seems as if the polyamines were displaced by other cations during the purification of the phage. When Brenner’s (7) permeable (osmotic-shock resistant) mutant of T4 was washed with Mg++, a preparation of phage was obtained containing essentially no polyamines; when the mutant phage was washed with spermidine and then with water, a balance was obtained between the DNA anions and the spermidine cations. The properties of preparations of T4 phage containing various cations have been examined.