The chapter focuses on the fractionation of transfer ribonucleic acid (tRNA) on columns of diethylaminoethyl (DEAE)-cellulose or DEAE-Sephadex at elevated temperatures that provides a simple procedure for large-scale fractionation of total tRNA or for further separation of tRNA fractions enriched for certain amino acid acceptor species by other methods. The hot column method takes advantage of differences in melting temperatures of various tRNA species, which arise from their different base sequences. Hot column method is used in two ways to fractionate tRNA. Fractionation is achieved at a constant temperature of 65° with a nonlinear salt gradient to elute the tRNA. The second method exploits a decreasing temperature gradient to elute the tRNA at a constant salt concentration. The choice of which of the two methods to use for any particular fractionation depends upon the species of tRNA that are under investigation. The importance of complete removal of ribonuclease from the tRNA prior to hot column chromatography must be emphasized. The fractionation achieved on hot DEAE columns presumably depends upon the degree of unfolding of the various tRNA species at the temperatures and salt concentrations employed. Hence, those tRNA's that are most unfolded are able to form the greatest number of bonds with the DEAE.