Various tRNA transcripts were constructed to study the identity elements of Escherichia coli tRNA(Asp). Base substitutions from G34 to U34 at the first position of the anticodon, and from U35 to A35 at the second, severely impaired the aspartate charging activity. The activity was also decreased, but in a more moderate fashion, by base changes at G2-C71, C36 and C38. Identity nucleotides of tRNA(Asp) are distributed in a different fashion between E. coli and yeast, which occur at the second base pair of the acceptor stem, G10-U25 base pair in the D-stem and 3' half of the anticodon loop.