A tRNA activator was isolated from mammalian organs [dog or rabbit liver] which increases the capability of tRNA to accept certain amino acids through the action of mammalian aminoacyl-tRNA synthetases. This activity may be separated from the aminoacyl-tRNA synthetases for isoleucine, leucine, lysine, serine and methionine by fractionation of liver or pancreas cytosol with ammonium sulfate or by chromatography over Sephadex G-200. The tRNA activating material is nondialyzable and is destroyed by trypsin or short heating. It acts catalytically. A MW of approximately 45,000 was obtained by chromatography of tRNA activator on a calibrated Sephadex G-150 column. Activator increases acceptance of yeast tRNA for the amino acids isoleucine, leucine, lysine, serine and methionine. It shows higher activity on liver tRNAMetf, tRNAMetm and tRNALys than on unfractionated liver tRNA. Removal of protein from mammalian tRNA by extra phenol extractions, chromatography or proteinase treatment increases its response to activator.