Abstract.-A sensitive method of polyamine estimation has been adapted to the study of the organic cations of small amounts of nucleic acid. A procedure utilizing phenol extraction, alcohol precipitation, and separation on Sephadex G100 has been devised for the isolation of tRNA at low ionic strength. The procedure is applicable to the isolation of tRNA from liter batches of bacterial culture. With these methods we have examined the polyamines of tRNA isolated from polyauxotrophic strains of E. coli incubated under various physiological conditions and have found the following: (1) The tRNA from relaxed bacteria (TAU rel) harvested during exponential growth is heterogeneous with respect to polyamine content. Some portions of the population contain about one mole of spermidine per mole of tRNA. Some putrescine and an unknown amine are also present in low concentration. (2) After exponential TAU rel is incubated with thymine and uracil in the absence of arginine, the tRNA population is far more homogeneous with respect to polyamine content. The various fractions contain two moles of spermidine per mole of tRNA and a small amount of putrescine. (3) After exponential TAU rel is incubated in the absence of both arginine and uracil, the polyamine pattern of the tRNA resembles that isolated from exponential cells. (4) The tRNA from stringent bacterias harvested during exponential growth is heterogeneous with respect to polyamine distribution and some fractions contain relatively high concentrations of the unknown amine.We have been studying the relationship of synthesis of RNA and of the polyamines in polyauxotrophic strains of E. coli.1, 2 In addition to known effects of spermidine on RNA synthesis in vitro,3 our observations have led us to conclude that it is a controlling element in RNA synthesis in vivo. Our data have shown (1) the concomitant synthesis in bacteria of RNA and of this organic cation in numerous physiological conditions,2 (2) the relaxation of synthesis of ribosomal RNA in stringent bacteria by exogenous addition of spermidine,1 and (3) the relatively high rate of synthesis of spermidine in a relaxed mutant as compared to the stringent parent, a difference expressed even in the absence of RNA accumulation.4 It may be asked whether these effects of spermidine arise mainly from the activation of transcription or by the reaction of this polyamine with the main RNA products, rRNA and tRNA.That spermidine can serve to organize helical structure in both RNA5 and DNA6 is known. Indeed, it has been shown that polyamines can help to effect a conversion in vitOm of the inactive configuration of tRNA to the active form.7'-Spermidine appears to be the most active organic cation in this conversion.10 Furthermore, spermidine can replace Mg++ in the methylation of tRNA by liver tRNA methylasell and in the transfer of an amino acid from a complex of enzymc-amino acid-adenylate to tRNA.12 In seeking to define a physiological 669