2′-Deoxynucleoside 5′-α-thiotriphosphates have been incorporated randomly, replacing any of the four nucleotides separately and at a low level in Escherichia coli tRNAAsp transcripts. After some tRNAs were charged with the cognate aminoacyl-tRNA synthetase and biotinylated, charged and uncharged tRNAs were separated by binding to Streptavidin. A comparison of the iodine cleavage pattern of charged and uncharged tRNAs indicated positions of 2′-deoxy-phosphorothioate interference with charging. To separate the 2′-deoxy from the phosphorothioate effect, the same sequence of reactions was performed with the corresponding NTPαS. Several positions were identified with a 2′-deoxy or a phosphorothioate effect. tRNAs with single deoxy substitutions at the identified positions were prepared by enzymatic ligation of chemically synthesized halves. The kinetics of charging these tRNAs were determined. The 2′-deoxy effects identified by the interference assay were confirmed, showing a reduction in charging efficiency of between 2.5–6-fold, except for the terminal A76 with a 25-fold reduction. Inspection of the X-ray structure of the tRNA-synthetase complex showed consistency of most of these findings. Critical 2′-deoxy groups are localized mainly on the proposed contact surface with the synthetase or at the interface of the two tRNA domains. The same overall picture emerged for critical phosphorothioates. With the exception of 2′-deoxy-adenosine-containing tRNAs, multiple 2′-deoxy-substituted tRNAs, prepared by ligation of halves, showed a much larger reduction in charging efficiency than the mono-substituted tRNAs, indicating an additive effect.