1. The sites within the tRNA sequence of nucleosides methylated by the action of enzymes from mouse colon, rat kidney and tumours of these tissues acting on tRNAAsp from yeast and on tRNAGlu2, tRNAfMet and tRNAVal1 from Escherichia coli were determined. 2. The same sites in a particular tRNA were methylated by all of these extracts. Thus tRNAGlu2 was methylated at the cytidine residue at position 48 and the adenosine residue at position 58 from the 5′-end of the molecule; tRNAAsp was methylated at the guanosine residue at position 26 from the 5′-end of the molecule; tRNAfMet was methylated at the guanosine residues 9 and 27, the cytidine residue 49 and the adenosine residue 59 from the 5′-end; tRNAVal1 was methylated at the guanosine residue 10, the cytidine residue 48 and the adenosine residue 58 from the 5′-end. 3. All of these sites within the clover leaf structure of the tRNA sequence are occupied by a methylated nucleoside in some tRNA species of known sequence. It is concluded that methylation of tRNA from micro-organisms by enzymes from mammalian tissues in vitro probably does accurately represent the specificity of these enzymes in vivo. However, there was no evidence that the tumour extracts, which had considerably greater tRNA methylase activity than the normal tissues, had methylases with altered specificity capable of methylating sites not methylated in the normal tissues.