Abstract We have determined the nucleotide sequences of 55 spontaneous mutations that inactivate a suppressor gene of phage T4 tRNA Ser. Most of the mutations caused substitutions or deletions of single nucleotides at 18 different positions in the tRNA. Two of three mutations that allowed the synthesis of mature tRNA had nucleotide substitutions at the junction of the dihydrouridine and anticodon stems, suggesting that this region of tRNA Ser is important for aminoacylation. The third mutation that synthesized tRNA had a nucleotide deletion in the anticodon loop, which presumably affected the translational capacity of the tRNA. We also sequenced 58 spontaneous reversion mutations derived from strains with the inactive suppressor genes. Some of these regenerated the initial tRNA sequence, while others generated a second-site mutation in the tRNA. These second-site mutations restored helical base-pairings to the tRNA that had been eliminated by the initial mutations. The new base-pairings involved G · C and A · U, and the A · C wobble pair at certain positions in the tRNA. This finding establishes the existence of A · C wobble pair in tRNA helices.