Suppressor tRNAs are useful tools for determining identity elements which define recognition of tRNAs in vivo by their cognate aminoacyl-tRNA synthetases. This study was aimed at the isolation of active amber tRNA(Asn). Nineteen mutated tRNA(Asn)CUA having amber suppressor activity were selected by an in vivo genetic screen, and all exclusively inserted glutamine. From analysis of the different mutations it is concluded that glutamine accepting activity was obtained upon reducing the interaction strength between the first base pair of the tRNA(Asn)CUA by direct or indirect effects. Failure to isolate tRNA(Asn)CUA suppressors charged with asparagine as well as other evolutionary related amino acids is discussed.