A transfer RNA complete devoid of modified nucleosides was synthesized by in vitro transcription, and some of its properties in aminoacylation and protein synthesis in vitro were studied. For this purpose, a plasmid was constructed which contained a glycine tRNA gene from Mycoplasma mycoides under the promoter of the T7 RNA polymerase, as well as a BstNI restriction site at the 3'-end of the tRNA gene. Cleavage of plasmid DNA with BstNI followed by T7 RNA polymerase transcription in vitro yielded an RNA which was processed with M1 RNA, the catalytic subunit of ribonuclease P, to give a tRNA of mature length. The tRNA synthesized in this manner can be esterified with glycine in vitro, and the rate of aminoacylation is the same as when using the corresponding fully modified glycine tRNA from M. mycoides. Furthermore, in protein synthesis in vitro, the tRNA lacking modified nucleosides was essentially as efficient as the corresponding normal glycine tRNA. However, the Escherichia coli extract used in our protein-synthesizing system introduced one modification, pseudouridine, into the in vitro-synthesized tRNA, and it cannot be excluded that this modification has an essential role in protein synthesis.