Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely D-tRNAPhe and D-tRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity.