Summary Studies with 32P-labeled tRNA indicate that intact tRNA is efficiently taken up into Novikoff hepatoma ascites cells in the presence of DEAE-dextran (0.1 mg/ml). The 32P-tRNA was taken up intact as demonstrated by DEAE-cellulose column chromatography and sucrose density gradient centrifugation. The formation of a DEAE-dextran-tRNA complex was demonstrated by hyperchromicity, sucrose density gradient centrifugation, and the lack of precipitation of the tRNA in the complex with ethanol. Although DEAE-dextran protects tRNA from intracellular RNases, it does not protect tRNA against high concentrations of pancreatic RNase. Inhibition of uptake of tRNA by the cells with either iodoacetate (IC50 = 2 × 10-2M) or azide (IC50 = 8 × 10-2M) demonstrated that both cell association and uptake of tRNA are energy dependent. To demonstrate that tRNA was intracellular and not simply adsorbed, control studies were made at 0° and zero time and by treatment of the cells with pancreatic RNase after the incubations. On the basis of these studies, approximately 25% of the total tRNA in the incubation mixture was cell associated at 120 min in the presence of DEAE-D. Of the cell associated tRNA, approximately 40% was intracellular.